A highly selective, sensitive and “turn-on” fluorescent sensor for the paramagnetic Fe3+ ion

Accepted Manuscript Title: A highly selective, sensitive and “turn-on” fluorescent sensor for the paramagnetic Fe3+ ion Author: Thanasekaran Nandhini Palanichamy Kaleeswaran Kasi Pitchumani PII: DOI: Reference:

S0925-4005(16)30202-7 http://dx.doi.org/doi:10.1016/j.snb.2016.02.054 SNB 19714

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

20-11-2015 3-2-2016 12-2-2016

Please cite this article as: Thanasekaran Nandhini, Palanichamy Kaleeswaran, Kasi Pitchumani, A highly selective, sensitive and “turn-on” fluorescent sensor for the paramagnetic Fe3+ ion, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.02.054 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A highly selective, sensitive and “turn-on” fluorescent sensor for the paramagnetic Fe3+ ion Thanasekaran Nandhinia, Palanichamy Kaleeswarana, and Kasi Pitchumania,b,* a

Department of Natural Products Chemistry, School of Chemistry, Madurai Kamaraj University,

Madurai – 625 021, India. b*

Centre for Green Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai.

Corresponding author: Tel.: +91 452 2456614; fax: +91 452 2459181. E-mail address: [email protected]

1

Abstract A

highly

selective

and

sensitive

chemosensor

for

Fe3+

ion

using

2-((4-methylbenzo-[d]thiazole-2-ylimino)methyl)phenol is developed. The probe 1 is found to be more selective towards Fe3+ over other metal ions such as Li+, Na+, K+, Ca2+, Mn2+, Cr3+,Co2+, Ni2+, Cu2+, Hg2+, Pb2+, Cd2+, Al3+, Zn2+, Ru3+and Ag+ with a limit of detection 0.89 nM. Upon binding to Fe3+ ion, a strong fluorescent enhancement is observed which is attributed to an increase in Charge Transfer. A 1:1 binding between probe 1 and Fe3+ ion is evidenced by Job’s plot. The proposed mechanism is strongly supported by TD-DFT calculations.

Keywords: Paramagnetic ion, Turn on Fluorescence, Sensor, Fe (III), Charge transfer, Chemosensor.

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Introduction Iron is the most essential metal ion for the proper functioning of all living cells and is mainly involved in oxygen metabolism and electron transfer processes in DNA and RNA syntheses. In a mitochondrial respiratory chain enzymatic reaction, iron acts as cofactor. Haemoglobin contains 3 to 4 g of elemental iron in adults. In the form of ferritin or hemosiderin iron is stored in liver, spleen and bone marrow of human body [1-2]. The deficiency of iron causes a disease namely anaemia and functional deficits associated with anaemia include gastrointestinal disturbances and impaired cognitive function, immune function, exercise or work performance, and body temperature regulation. In infants and children, iron deficiency can result in psychomotor and cognitive abnormalities that, without treatment, can lead to learning difficulties [3]. Acute intake of more than 20 mg/kg iron from supplements or medicines can, however, lead to gastric upset, constipation, nausea, abdominal pain, vomiting and faintness, especially if food is not taken at the same time. Hemochromatosis, a disease caused by a mutation in the hemochromatosis (HFE) gene, is associated with an excessive build-up of iron in the body. Overdoses of iron can lead to multisystem organ failure, coma, convulsions and even death [4-5]. However, both its deficiency and excess from the normal permissible limit can induce serious disorders. There are several analytical techniques such as AAS, [6-7], voltammetry [8] and potentiometry [9] are available for the sensing of Fe3+ ions. However, these available analytical methods are expensive, time-consuming and require pre-treatment experiments. Among the various techniques used for its detection, fluorescent sensors have received considerable interest in recent years because of their ability to provide online monitoring of very low concentrations without any pre-treatment of the sample together with the advantages of spatial and temporal resolutions.

3

Recently many iron sensors have been developed using spectrometric [10], electroanalytical [11], potentiometric [12], rhodamine based [13-18], quantum dot based [1920], methods

and also using nanoparticles [21-22]. However, successful development of

fluorescent sensors for iron is challenging due to its paramagnetic nature, which involves fluorescent quencher. Consequently considerable attention has been focused in recent years for the development of simple, selective and inexpensive sensor for iron. Our interest in developing chemosensors for biologically important cations [23-30] anions [31-33], and neutral molecules [34-35] prompted us to develop a simple, selective, sensitive and fluorescence chemosensor involving probe 1 for the sensing of Fe3+ ion. The present system is very simple, cost effective and sensitive towards Fe3+ ion which is employed in the eco-friendly aqueous medium.

Materials and Methods Chemicals: All chemicals used were of analytical grade and used without further purification. 2Amino-4-methylbenzothiazole was purchased from Aldrich. Salicylaldehyde and all the metal salts (LiCl, NaCl, KCl, CaCl2.2H2O, MnCl2.4H2O, CrCl3.6H2O, CoCl2.6H2O, NiCl2. 6H2O, CuCl2.2H2O, HgCl2, FeCl3.6H2O, PbCl2, CdCl2, AlCl3, ZnCl2, RuCl3, AgNO3,) were

purchased from Merck. UV-Vis absorption spectra were recorded by using the JASCO Spectra Manager (V-550) in a 1 cm path length quartz cuvette. All fluorescence measurements were performed on a Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon) with excitation slit set at 5.0 nm band pass and emission at 5.0 nm band pass in a 1 cm quartz cell. Electrospray ionization mass spectrometry (ESI-MS) analysis was performed in the positive and negative ion modes 4

on a liquid chromatography–ion-trap mass spectrometry instrument (LCQ Fleet, Thermo Fisher Instruments Limited, USA). The samples were introduced into the ion source by the infusion method at flow rate 1 mL min-1. The capillary voltage of the mass spectrometer was 33 V, with source voltage 4.98 kV for the mass scale (m/z 50–400). DFT calculations were performed at the B3LYP/LANL2DZ(d) level by using the Gaussian 05 program. Characterization details: 1H

NMR (300 MHz, CDCl3) (δ) 12.27 (s, 1H), 9.23(s, 1H), 7.65 (t, 1H, J=9HZ), 7.52 (d, 1H,

J=1.5 HZ), 7.45 (d, 1H, J=1.8 HZ), 6.96-7.07 (m, 4H), 2.73 (s, 3H, CH3) (Figure S1). 13C

NMR (75 MHz, CDCl3) (δ) 167.73, 167.16, 161.85, 150.83, 135.19, 134.48, 133.96,

133.23, 127.17, 125.18, 119.68, 119.06, 118.39, 117.62, 18.37. (Figure S2). ESI - MS [m/z] (rel. Int.) (+Ve Mode) [269.14 ([M+H]+, 100.0%)] (Figure S3). Preparation of UV and fluorescence titrations: All the measurements were carried out in double distilled water which is free from ions. The stock solution of probe 1 (1 × 10−3 M) was prepared by dissolving probe 1 in 100 mL ACN:Water (v/v, 1:1) mixture. Solutions (1 × 10−3 M) of Li+, Na+, K+, Ca2+, Mn2+, Cr3+ ,Co2+, Ni2+, Cu2+, Hg2+, Pb2+, Cd2+, Al3+, Zn2+, Ru3+, Ag+, and Fe3+ ions were prepared by dissolving them in double distilled water. For UV and fluorescence titrations, probe 1 (1 mL of stock), and cations (0.1–1.0 mL of stock) were taken and analysed at room temperature.

Results and Discussion Synthesis of 2- ((4-methylbenzo[d]thiazole-2- ylimino)methyl)phenol

Scheme 1 2-Amino-4-methylbenzothiazole (2 mmol) was dissolved in 10 ml of ethanol and salicylaldehyde (2 mmol) and acetic acid (0.08 mmol) were added to the above solution. The

5

mixture was refluxed for 24 hrs. After completion of the reaction, the reaction mixture was neutralized with ammonium chloride solution (20%) and extracted twice with dichloromethane. The organic layer was washed with deionised water and then dried over anhydrous sodium sulfate. The filtrate was concentrated under vacuum and the residue was purified by column chromatography using silica gel (60-120 mesh), petroleum ether and ethyl acetate mixture (20%) and an yellow solid was obtained (76% yield). Selective Sensing of Fe3+ ion Probe 1 contains ring nitrogen and sulphur, imine nitrogen and hydroxyl group, which will provide a good platform for binding metal ions. The Absorption and emission spectrum of probe 1 only was analysed (figure S4 & S5). The cation-binding ability of probe 1, was studied by absorption and fluorescence titrations with various cations, such as Li+, Na+, K+, Ca2+, Mn2+, Cr3+,Co2+, Ni2+, Cu2+, Hg2+, Pb2+, Cd2+, Al3+, Zn2+, Ru3+ and Ag+ in ACN:water (1:1) mixture. Excepting Fe3+ ion, all other metal ions show no response and the details are given in (figure 1).

Fig. 1 Fluorescent titration of Fe3+ ion Addition of most of the metal ions shows no response in fluorescence titration with probe 1, excepting the case of Fe3+. The sensitivity of Fe3+ detection by probe 1 is also studied in ACN:water (1:1) mixture. The concentration of Fe3+ is varied from 5 x 10-7 to 5 x 10-4 M. Due to the presence of binding sites in probe 1, Fe3+ binds with it effectively even at very low concentration and enhances the emission intensity with a blue shift. Free Probe 1 shows two emission bands at 430 and 574 nms. Upon addition of Fe3+ ion, the intense peak at 430 nm is blue shifted and an enhanced emission peak centred at 482 nm is observed.

6

The fluorescence intensity increased linearly with an increase in the concentration of Fe3+ ion. The value of the linearly dependent coefficient (R2) was found to be 0.99 and the limit of detection was 0.89 nM (3.3σ slope-1). The binding constant value was found to be 3.6 x 106 M-1, which indicates a stronger binding between probe 1 and Fe3+ ion (figure 2). The binding between probe 1 and Fe3+ ion was also confirmed by Job’s plot, from the absorption data, which is in good agreement with a 1:1 stoichiometry.

Fig. 2

Binding between probe 1 and Fe3+ ion is also evident from ESI – MS data. Initially a peak at m/z = 269 .14 (M + H)+ value is observed which corresponds to probe 1. When one equiv. of Fe3+ ion is added, the peak at m/z = 269.14 disappears and a new peak corresponding to [probe 1 + Fe3+ -H ion] appears at m/z = 427.97 (figure 3).

Fig.3

Competitive binding study of probe 1 The binding behaviour of probe 1 towards Fe3+ and other metal ions was also investigated. A competitive binding assay was performed in the presence of other metal ions(Li+, Na+, K+, Ca2+, Mn2+, Cr3+,Co2+, Ni2+, Cu2+, Hg2+, Pb2+, Cd2+, Al3+, Zn2+, Ru3+ and Ag+) with Fe3+ and probe 1 (figure 4). Interestingly, probe 1senses Fe3+ ion selectively even in the presence other metal cations in the mixture. While adding other paramagnetic metal ions such as Cu2+ and Co2+, a significance quenching was observed which is due to the quenching pathway involving low-lying d–d states, whereas in the presence of Fe3+ ion, no quenching of fluorescence is observed even in the presence of Cu2+ and Co2+ ions. This result 7

indicates that even in the presence of other paramagnetic cations, probe 1 can be successfully employed to selectively bind Fe3+ ion with fluorescence enhancement.

Fig. 4 Effect of pH The influence of pH plays a significant role in sensing metal cations and their biological applications. The present study indicates that effective binding is observed in the pH range of 6 to 7. At low pH, there is competition between H+ and Fe3+ ions leading to no binding of Fe3+ ion with probe 1. Beyond pH 7, negligible effect was observed in the fluorescence intensity (figure 5).

Fig. 5 DFT calculations The geometries of probe 1 and its complex with Fe3+ ion were optimised using DFTB3LYP6-31G and LANL 2DZ (d) levels respectively using Gaussion 05 package. As shown in (figure 6), the optimised geometry of the sensor probe 1 shows effective binding sites to form a 1:1 complex with Fe3+ ion and this supports the experimental findings obtained from Job’s plot in analysis of the complex (S6).

Fig. 6 To get an insight into the electronic behaviour in the presence and absence of Fe3+ ions with probe 1, DFT calculations were carried out. Plots of HOMO and LUMOs of probe 1, given in (figure 7), show that in probe 1, HOMO is localised on thiazole moiety and

8

LUMO at imine group. Upon addition of Fe3+ ion, HOMO is shifted to imine group and LUMO at Fe3+ ion, indicating substantial charge transfer.

Fig. 7

Cell imaging HeLa cells were grown in well plates in Dublecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (fetal bovine serum) at 37 oC. The HeLa cells were incubated with the probe 1 (5.0 µM in DMSO/H2O (2:1, v/v) in PBS) for 30 minutes and imaged through the microscope. After washing with PBS buffer for three times to remove the excess probe 1 in the extracellular parts of the cells, the cells were further incubated with Fe3+ (20.0 µM in H2O) for 10 minutes at 37°C and imaged with Olympus

fluorescence

microscope. The probe 1 treated cells did not show any fluorescence in cellular regions upon addition of FeCl3, enhanced fluorescence was observed in intracellular regions of cells. The bright field images clearly showed the cells are viable throughout the experiments (Figure 8). The intensity of probe 1 and Fe3+ ion with HeLa Cell Fluorescence imaging was studied (Figure S7). The values of Corrected Total Cell Fluorescence (CTCF) for probe 1 + HeLa cell and probe 1 + HeLa cell + Fe3+ ion are 1636.827 and 44450.84, given in Table 1. We have used the following formula to calculate the Corrected Total Cell Fluorescence. CTCF = Integrated Density - (Area of selected cell X Mean fluorescence of background readings)

Fig 8

9

Mechanism of Fe3+ selective sensing Upon addition of Fe3+ ion to probe 1, an increase in fluorescence intensity of probe 1 is observed. A blue shift is also observed at 482 nm after adding 5x10-6 M of Fe3+ ion to probe 1. Upon increasing the concentration of Fe3+ ion, the peaks at 430 and 574 nms are merged and a new peak centred at 482 nm has appeared. This may be due to the decrease in charge transfer from thiazole moiety to imino moiety, which is arrested while adding Fe3+ ion to probe 1. On the other hand, the presence of positively charged Fe3+ ion facilitates an increase in charge transfer from imino group to Fe3+ ion (figure 9).

Fig. 9

Comparison with previously reported methods The sensing ability of the present probe 1 towards Fe3+ ion was also compared with other

reported literature methods, which is given in (Table 2). It is evident, from the

comparison, that the present probe 1 is a cost effective and simple sensor for Fe3+ ion and its LOD is superior to the potentiometric methods, colorimetric methods and nanoparticles reported earlier. The present method involves a simple, highly sensitive, selective method and avoids the need for sophisticated and costly instruments.

Table 2

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Conclusions A 2-((4-methylbenzo-[d]thiazole-2-ylimino)methyl)phenol moiety is developed as a simple and selective “turn on” chemosensor for Fe3+ ion based on an Intramolecular Charge Transfer mechanism at the nanomolar level in ACN - water mixture. The probe 1 is more selective towards Fe3+ ion in the presence of other metal ions, such as Li+, Na+, K+, Ca2+, Mn2+, Cr3+, Co2+, Ni2+, Cu2+, Hg2+, Pb2+, Cd2+, Al3+, Zn2+, Ru3+ and Ag+, with a limit of detection 0.89 nM. Probe 1 forms 1:1 complex with Fe3+ ion with a binding constant value of 3.6 x 106 M-1 indicating that stronger binding occurs between probe1 with Fe3+ ion, which is also evident from Job’s plot. A strong binding of Fe3+ ion with the hydroxyl group, imino group and thiazole nitrogen is proposed, which results in an enhanced fluorescence. An charge transfer mechanism is operating which consequently results in a blue shift and fluorescence enhancement. A suitable mechanism is proposed to rationalise the observed results. The probe 1 senses Fe3+ ions effectively at a pH range of 6-7 in aqueous solution, which is very suitable for biological studies. The proposed binding mode between probe 1 and Fe3+ ion, and the fluorescence behaviour are explained from TD-DFT studies.

Acknowledgments T.N., P.K. and K.P. gratefully acknowledge, CSIR and UGC-UPE, New Delhi.

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Biographies Thanasekaran Nandhini (1990) received her BSc (Chemistry) degree from Devanga Arts College (Madurai Kamaraj University), Aruppukottai in 2010 and MSc (Chemistry) from V.H.N.S.N College (Madurai Kamaraj University), Virudhunagar in 2012. At present she is a Research Scholar in Madurai Kamaraj University. Her research focuses on biosensor, quantum dot based sensor and nanomaterials. Palanichamy Kaleeswaran (1989) received his BSc (Chemistry) degree from Ayya Nadar Janaki Ammal College, Sivakasi in 2009 and MSc (Chemistry) from Ayya Nadar Janaki Ammal College, Sivakasi in 2011. At present he is a Senior Research Fellow in Madurai Kamaraj University. His research focuses on biosensor, chemosensor and supramolecular based sensor Kasi Pitchumani (1954) received his M.Sc. (Chemistry) from Madurai Kamaraj University, Madurai, India. He has received Ph.D., degrees from the same university in 1981 and was appointed as Professor in Organic Chemistry from 1996 to till present. He has received D.Sc. on from Madurai Kamaraj University. He did his postdoctoral research with Prof. V. Ramamurthy, University of Miami, USA and Prof. Akihiko Ueno, Tokyo Institute of Technology, Japan. He has 35 year of teaching experience in Organic Chemistry and published 181 research articles in peer reviewed journals. His research interests are supramolecular photochemistry and chemistry in confined media like clays, zeolites, hydrotalcites, cyclodextrins, synthesis of modified cyclodextrins, isolation of natural products and newer nanomaterials for developing sensor applications.

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Figure captions:

Figure 1: A) Fluorescence spectra of probe 1 (1x10-5 M) on addition of (Li+, Na+, K+, Ca2+, Mn2+, Cr3+, Co2+, Ni2+, Cu2+, Hg2+, Pb2+, Cd2+, Al3+, Zn2+, Ru3+, Ag+) and Fe3+ ions (1x10-5 M). λexc = 380 nm. B) UV-Vis spectra of probe 1 (1x10-5 M) in the presence of Fe3+ and various metal ions (Li+, Na+, K+, Ca2+, Mn2+, Cr3+, Co2+, Ni2+, Cu2+, Hg2+, Pb2+, Cd2+, Al3+, Zn2+, Ru3+, Ag+) (1x10-5 M) at pH 7 Figure 2: Fluorescence response of Probe 1 on addition of Fe3+ (5x10-7- 5x10-4 M). The insert shows a linear fit in the plot of intensity vs concentration of Fe3+ ion Figure 3: ESI–MS spectrum of 2- ((4-methylbenzo[d]thiazole-2- ylimino)methyl)phenol with Fe3+ ion (probe 1 + Fe3+ ion) Figure 4: Bar chart showing the fluorescence selectivity of probe 1 (5x10-7 M) towards Fe3+ ion in the presence of other metal ions (5x10-7 M). The red bars represent the fluorescence intensity of probe 1 in the presence of one equivalent of other metal ions. The green bars represent the fluorescence intensity of one equivalent of probe 1 containing one equivalent of other metal ions and Fe3+ ions Figure 5: Influence of pH on fluorescence titration of probe 1 with Fe3+ ion Figure 6: Proposed optimised geometry of probe 1 with Fe3+ Figure 7: Frontier molecular orbitals optimized at the 3LYP/LANL2DZ(d)level of theory Figure 8: cell imaging of Fe3+ in HeLa cells: A) bright-field image of probe 1 treated HeLa cell B) fluorescence image of HeLa cells incubation with probe 1 (5 μM) for 30 min at 37 ◦C

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C) fluorescence image of HeLa cells incubated with probe 1 (5 μM) and subsequently treated with FeCl3 (20 μM) for 10 min at 37oC Figure 9: Mechanism of Fe3+ ion detection

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Fig 1

23

Fig 2

24

Fig 3

25

Fig 4

26

Fig 5

27

Fig 6

28

Fig 7

29

Fig 8

30

Fig 9

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Scheme 1: Synthesis of 2- ((4-methylbenzo[d]thiazole-2- ylimino)methyl)phenol

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Table 1: CTCF values of probe 1 and Iron ion from cell imaging S. No

Name

CTCF

1

Probe 1 + HeLa Cell

1636.827

2

Probe 1+ HeLa Cell + Iron

44450.84

Table 2: Comparison with previous literature reports S. No

Probe

1 2

10

Polyphenyl derivative Poly (propyleneamine) dendrimer 1,3,4-Oxadiazole and phosphonic acid Coumarin derivative Carbazole-based Schiff-base 1,10-(4-Methylbenzene-1,3diyl)bis[3-(2sulfanylphenyl)urea] Diaza-18-crown-6 ether appended with dual coumarins Rhodamine-benzimidazole Conjugate 2-(2Hydroxyphenyl)benzothiazole Rhodamine based triazole

11 12 13 14

3 4 5 6

7

8 9

15 16

Method

Biological Studies ---

Reference

Fluorescence Fluorescence

Limit of detection 4.0x10-6 M 2x10-7 M

Fluorescence

1x10-8 M

--

[39]

Colorimetric Fluorescence Fluorescence

0.09l M 5.67x10-6 0.1l M

----

[40] [41] [42]

Fluorescence

0.31 μM

--

[43]

Fluorescence

1.5x10-8 M

--

[44]

Fluorescence

6.04x10-8 M

--

[45]

Fluorescence

5.0x10-8 M

[46]

Spirobenzopyran-quinoline Chalcone Rhodamine B derivative

Fluorescence chromo/fluorogenic Fluorescence

1x10-8 M 5.8x10-8 M 0.418 ppm

1,8-naphthalimide-based fluorescence chemosensor 2-methoxy-6-((quinolin-8ylimino)methyl)phenol This method

Fluorescence

2 μM

Fluorescence

1.3x10-7 M

NIH 3T3 cells living cells -EC 109 cells MDA-MB231 cells HeLa cells

Fluorescence

8.9x10-8 M

HeLa cells

[37] [38]

[47] [48] [49] [50] [51]

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