Reversible NIR fluorescent probes for Cu2+ ions detection and its living cell imaging

Accepted Manuscript Title: Reversible NIR Fluorescent probes for Cu2+ ions detection and its Living cell imaging Authors: Gujuluva Gangatharan Vinoth Kumar, Mookkandi Palsamy Kesavan, Gandhi Sivaraman, Jamespandi Annaraj, Kandasamy Anitha, Arunachalam Tamilselvi, Shenmuganarayanan Athimoolam, Balasubramaniyam Sridhar, Jegathalaprathaban Rajesh PII: DOI: Reference:

S0925-4005(17)31816-6 http://dx.doi.org/10.1016/j.snb.2017.09.150 SNB 23241

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

31-7-2017 15-9-2017 21-9-2017

Please cite this article as: Vinoth Kumar Gujuluva Gangatharan, Kesavan Mookkandi Palsamy, Sivaraman Gandhi, Annaraj Jamespandi, Anitha Kandasamy, Tamilselvi Arunachalam, Athimoolam Shenmuganarayanan, Sridhar Balasubramaniyam, Rajesh Jegathalaprathaban, Reversible NIR Fluorescent probes for Cu2+ ions detection and its Living cell imaging, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.09.150 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.

Reversible NIR Fluorescent probes for Cu2+ ions detection and its Living cell imaging Gujuluva Gangatharan Vinoth Kumara,b, Mookkandi Palsamy Kesavana,b, Gandhi Sivaramanc*, Jamespandi Annarajd, Kandasamy Anithae, Arunachalam Tamilselvif, Shenmuganarayanan Athimoolamg, Balasubramaniyam Sridharh, Jegathalaprathaban Rajesha,b* a

Department of Chemistry, Sethu Institute of Technology, Kariapatti-626 115, Tamilnadu, India.

b

Chemistry Research Centre, Mohamed Sathak Engineering College, Kilakarai-623 806, Tamil

Nadu, India. c

Institute for stem cell biology and regenerative medicine, Bangalore-560065, Karnataka, India.

d

School of Chemistry, Department of Material Science, Madurai Kamaraj University, Madurai-

625 021, Tamilnadu, India. e

f

School of Physics, Madurai Kamaraj University, Madurai-625 021, Tamilnadu, India.

Department of Chemistry, Thiagarajar College, Madurai-625 009, Tamilnadu, India.

g

Department of Physics, University College of Engineering, Anna University, Nagercoil-629

004, Taminadu, India. h

Laboratory of X-ray Crystallography, Indian Institute of Chemical Technology, Hyderabad-500

007, India. * Corresponding author *E-mail address: [email protected] (Dr. J. Rajesh) [email protected]( Dr. G. Sivaraman)

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GRAPHICAL ABSTRACT

Highlights



Efficient Schiff base probes 1 and 2 was synthesized and used to detect Cu2+ ions.



The detection limit of Cu2+ (0.22 μM and 0.729 μM) for 1 and 2 is reported.



DFT calculation was supported the experimental data and sensing mechanism. 2



These probes are successfully applied to molecular logic circuits.



Probes 1 and 2 are effectively concerned for real water samples.



Live cell imaging in HeLa cells were studied.

Abstract A new, easily accessible “off-on-off” colorimetric and fluorescent probes are designed and synthesized. It displayed highly selective and sensitive detection towards Cu2+ ion without having any interference from other competitive metal ions. The examined UV-Vis and fluorescence spectral changes are caused by the operation if the ligand to metal charge transfer (LMCT) and chelation enhanced fluorescence effect (CHEF) in the presence of Cu2+ to the present probes. Besides, the density functional theory (DFT) calculations have been theoretically supported the photophysical changes. These probes are potentially applied in an analytical application for detecting the trace amount of Cu2+ ions in real water samples. Furthermore, these probes are successfully applied for fluorescence imaging of Cu2+ in HeLa cells. Finally, the use of test strips based on these probes is fabricated, which could act as a convenient and efficient Cu2+ test kit in solution medium and supported on solid medium (silica). Key words: Copper ion sensors, turn-on fluorescence, Logic gates, DFT calculations, Live cell imaging.

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1. Introduction Fluorescence based chemosensors for metal ions has been great attention in modern years due to their assist in environmentally observing by their capability to detect various metal ions, even in very low concentration and also applied in medical research as imaging agents. Copper is the heavy metal and the third most abundance metal ion (after Fe3+ and Zn2+) in human bodies, plays a very essential role in various physiological process include bone formation, cellular respiration, connective tissue development and provides a major catalytic co-factor for several metalloenzymes [1-8]. If the remarkable level in copper ions can cause oxidative stress and neurological disorders like Alzheimer’s, Parkinsons, Menkes, and Wilson diseases [9-11]. Conversely, high concentration of copper is toxic and harm to the environment. Hence, it is necessary to develop highly sensitive and selective techniques that would be extremely useful for the real-time observation of the copper ions in environmental and biological samples [12-18] especially for the trace amounts of copper ion in real sample investigations. In recent years, the development of fluorescent sensors for copper ions with highly selectivity towards other metal ions have been successfully reported [19-21]. Various analytical methods such as photometric methods, inductively coupled plasma emission or mass spectrometry (ICP-ES, ICP-MS), atomic absorption spectroscopy (AAS), anodic stripping voltammetry (ASV) and total reflection Xray fluorimetry (TXRF) [22–24] involve a tedious procedure and desires high cost instrumentation. Whereas, colorimetric and fluorescence based techniques are preferable due to the low cost and

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easily concerned practice for the detection of copper ions. Moreover, naked-eye detection require no instrumentation. While, absorbance and fluorescence based sensing involves instrumentation make feasible bio-imaging of analytes and hence serve as diagnostic tool in medicinal field. In view of this, it is fascinating to design a chemosensor that detect copper ions, both by colorimetric and fluorescent modes. Nowadays the molecular identification analysis can be carried our with the help of information technology such as logic gates [25-27], information storage device [28], molecular keypad locks [29] and so on. Such logic instruments are considered to transfer the molecular level information to the examined optical signals [30-32]. The current interest is building of molecular logic circuits are capable of performing some special arithmetic operations are based on those showing more than one output signal with single molecule towards various logic functions [33, 34]. Besides, various valuable integrated logic gates such as INHIBIT, IMPLICATION, half adder, half subtractor, full adder and full subtractor have been developed with various single molecules [35]. Nevertheless, concerning IMPLICATION logic gates, which are corresponding to the IF-THEN operation and NOT operation, the researcher has rarely been reported [36]. In this work, we report two new fluorescent probes 1 and 2 by combining 5-chloro salicylaldehyde with 2-phenoxy aniline and 2-phenylthio aniline into a simple molecular framework (Scheme 1) for the selective detection of Cu2+ in PBS buffered solution. These probes are able to sense Cu2+ ions in the presence of various metal ions was observed by absorption and fluorescence spectrometry. To the best of our knowledge, detection event triggers fluorescence-on response to the probes 1 and 2 displays a prominent selectivity for Cu2+ ion

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towards other tested metal ions. Moreover, probes 1 and 2 were also applied for fluorescence imaging of Cu2+ in HeLa cells. (Scheme 1) 2. Experimental 2.1. Materials and methods 2-phenoxy aniline, 2-phenylthio aniline, 5-chloro salicylaldehyde and metal cations as their corresponding chloride salts such as Cu2+, Fe2+, Co2+, Ni2+, Zn2+, Mn2+, Mg2+, Pb2+, Ag+, Na+, Ca2+, Ba2+, K+ and analytical grade solvents are purchased from Universal scientific appliances, Madurai, Tamilnadu, India. The 1H NMR (400 MHz) spectra and 13C NMR (100 MHz) spectra recorded on a Bruker Avance spectrometer by using CDCl3 as solvent. IR spectrum performed on a FT-IR spectrometer (KBr discs) in the range of 4000-400 cm-1. The UV-Vis spectral measurements were carried out in UV-8453 Agilent spectrometer. Fluorescence measurements were recorded on a JASCO FP-6300 fluorescence spectrophotometer. The elemental analysis was determined on a Heraeus CHN rapid analyzer. Melting point (uncorrected) data was measured on Electrothermal 9200 instruments. Electro spray Ionization mass spectral (ESI-MS) analysis was recorded in the positive ion mode on a liquid chromatography-ion trap mass spectrometer (LCQ Fleet, Thermo Fisher instruments limited USA). Density Functional Theory (DFT) calculations were carried out with LANL2DZ basis set using Gaussian 09 program to evaluate the interaction mode of probes and optimized geometries of probes and Cu2+ were optimized by DFT-B3LYP using LANL2DZ basis sets. 2.2. Stock solution preparation for spectral detection

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The chloride salts of Cu2+, Fe2+, Co2+, Ni2+, Zn2+, Mn2+, Mg2+, Pb2+, Ag+, Na+, Ca2+, Ba2+, K+ were prepared in double distilled water as a stock solution (1 mM). The probes 1 and 2 stock solution (1 mM) were prepared in PBS buffer solution containing 1% DMSO (pH = 7.54, 500 μM). The working solutions of 1 and 2 were freshly prepared by diluting the highly concentrated stock solution to the preferred concentration preceding to spectroscopic measurements. 2.3. UV-Vis and Fluorescence spectroscopic methods The UV-Vis and fluorescence spectral response of the probes 1 and 2 towards various metal ions were examined by absorption and fluorescence spectroscopy in PBS buffer solution. Emission spectrum recorded at a scan rate of 500 nm/min and the excitation wavelength of 320 nm for 1 and 2. Each experiment was recorded at least twice to attain the reliable values. 2.4. Application in real water samples The potential applicability of the probes 1 and 2 to detect Cu2+ ion in three real water samples was determined using spikes and recovery method. Tap water sample was attained at our laboratory water pipe and the sewage water sample was acquired at our college of Sethu Institute of Technology patio. Whereas, the third sample was natural water samples taken from vaigai river, Madurai. All the collected samples are simply filtered. Then all the water samples are spiked with a standard solution of Cu2+ ion (final concentration, 12 μM) and then its absorption intensity changes were examined before and after being spiked with different concentration of Cu2+ ion was performed with proposed probes via absorption spectroscopic method. 2.5. Cell culture and Fluorescence imaging HeLa cells were developed in modified Eagle's medium supplemented with 10% FBS (fetal bovine serum) at 37° C. The HeLa cells were incubated with probes 1 and 2 (5.0 µM) in 7

PBS buffer, pH = 7.54, containing 1% DMSO as co-solvent , after incubation they cells are washed with buffer for thrice to eliminate excess of probes present in the extracellular media and growth medium. The cells were imaged through the fluorescence microscope. Again the probes treated cells were further incubated with CuCl2 (5.0 µM in H2O) for 10 min at 37°C and imaged with a fluorescence microscope. 2.6. Synthesis of colorimetric receptors 2.6.1. Synthetic protocol of 1 The probe 1 was synthesized by condensing 2-phenoxy aniline with 5-chloro salicylaldehyde. Briefly, 2-phenoxy aniline (0.2g, 1.08 mM) and 5-chloro salicylaldehyde (0.17g, 1.08 mM) was dissolved in methanol and stirred for 3 hours at room temperature. The completion of the reaction was examined by TLC plate for the vanishing of starting compounds. After the completion of the reaction slow evaporation of the solvent under the same condition to give the brown colored powder. The acquired powder was washed with methanol and dried in anhydrous CaCl2. Yield: 65.4%. Melting Point: 95°C. 1H NMR (400 MHz, CDCl3, δ, ppm), (Fig. S1a): 13.11 (s, 1H), 8.61 (s, 1H), 7.31- 7.30(m, 3H), 7.28-7.27 (m, 2H), 7.24-7.23 (m, 2H), 7.08-7.02 (m, 2H), 6.98-6.95 (m, 2H),6.89-6.85 (d, 1H, J- 1.6Hz). 13C NMR (100 MHz, CDCl3, δ, ppm), (Fig. S1b): 162.1, 160.2,157.2, 150.1, 133.3, 131.5, 130.2, 128.8, 125.1, 123.8,123.5,, 121.3, 120.7, 120.4. Elemental analysis: C19H14NO2Cl, Found (calcd. %): C, 69.82 (70.26); H, 5.60 (5.54); N, 8.9 (9.2). 2.6.2. Synthetic protocol of 2 The probe 2 was synthesized by adopting the same procedure of 1. 0.2g (0.994 mM) of 2-phenylthio aniline and 0.156g (0.994 mM) of 5-chloro salicylaldehyde in methanol were charged in RB flask and the contents were stirred for 30 minutes and the completion of the 8

reaction was monitored by TLC. Then the obtained yellow solid was filtered and swabbed with methanol and dried in vacuo over anhydrous CaCl2. Yield: 62%. Melting Point: 110 °C. 1

H NMR (400 MHz, CDCl3, δ, ppm), (Fig. S2a):13.06 (s, 1H), 8.51 (s, 1H), 7.41- 7.33(m, 2H),

7.31- 7.29(m, 6H), 7.29- 7.22 (m, 2H), 7.18- 7.15(m, 2H), 7.14-7.12 (d, 1H, J=1.6 Hz). C NMR (100 MHz, CDCl3, δ, ppm), (Fig. S2b): 161.4, 160.0, 133.5, 133.1, 132.9. 131.65,

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131.62, 129.9, 129.3,128.2, 127.9, 123.9, 120.3, 119.4, 118.3. Elemental analysis: C19H14NOSCl, Found (calcd. %): C, 71.48 (71.53); H, 4.23 (4.3); N, 9.76 (9.79). ESI-MS for C19H14NOSCl (M+H+): Calcd, 339.839: found, 340.35. 2.7. Crystallographic data collection and refinement The compound 2 was crystallized from solvent evaporation solution growth method. Block type single crystals suitable for X-ray diffraction were chosen from the grown sample. The crystallographic data collection, using the X-ray with wavelength of 0.71073 Å, was collected at room temperature with MoK radiation using Bruker AXS KAPPA APEX-2 diffractometer equipped with graphite monochromator [37]. The structure was solved by direct methods and refined by full-matrix least-squares calculations using SHELXL-2014 [38, 39]. All the H atoms pertained to the carbon atoms were placed geometrically calculated position (-CH = 0.93 Å) and constrained to ride on their parent atoms with Uiso(H) = 1.2Ueq (parent atom). The H atom pertained to the oxygen atom is located from the difference Fourier map and refined isotropically. The crystallographic data, details of data collection and the structure refinement are given in Table S1. The ORTEP view of the molecules drawn at 50% probability thermal displacement ellipsoids with the atom numbering scheme is shown in Fig. 1. X-ray crystallographic information data (CIFs) is available with the CCDC deposition with number 1530024 for this paper. These data can be acquired via 9

http://www.ccdc.cam.ac.uk or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, U.K.; Fax: +44 1223 336033. (Figure 1)

3. Results and discussion 3.1. Molecular geometry and crystal packing of compound 2 The orthorhombic centrosymmetric lattice of compound 2 contains eight units of the asymmetric part in the unit cell. The convergent of the structure solution is confirmed from the R factor of 3.16 %. Three aromatic rings of the compound are oriented with angles of 32.69(6) [between C1--C6 and C9--C14 rings], 80.46(6) [between C9--C14 and C15--C20 rings] and 72.53 [between C15--C20 and C1--C6 rings]. Further, these aromatic rings, viz., C1--C6, C9-C14 and C15--C20, are oriented with angles of 89.16(4), 79.81(5) and 37.14(4), respectively. The hydrogen bonding dimensions are listed in Table S2 and the packing arrangement of molecules is represented in Fig. 2a. Intermolecular interactions, especially classical and nonclassical hydrogen bonds, are playing a crucial role in the formation of crystalline solids and their physiochemical properties [40]. These hydrogen bonding interactions can be classified and notated with graph-set nomenclature which is useful in comparing the stability of the interactions [41]. The molecular structure stabilized through intramolecular O-H...N and O-H...S interactions, which are designated with self associated S(6) and S12(5) motifs. Moreover, the crystal packing is stabilized during O-H...O, C-H...O and - interactions. The expected para chlorine is not participating in the intermolecular interactions. The self associated ring motifs, viz., S(6) and S12(5), were connected through a O-H...S hydrogen bond leading to centrosymmetric ring 10

R22(14) motif. These ring motifs are connected along the a-axis of the unit cell forming chain C22(7) and C22(8) motifs. Further, the aromatic rings of C1--C6 are stacked face-to-face in crystalline lattice, leading to - interactions which further support the molecular assembly along b-axis of the unit cell. The hydrogen bonding networks with the necessary graph-set notations are illustrated in Fig. 2b. (Figure 2a) (Figure 2b) 3.2. Colorimetric sensing property Colorimetric detection by naked-eye is the simplest method to recognize the selectivity of probes towards the various tested metal ions (Cu2+, Fe2+, Co2+, Ni2+, Zn2+, Mn2+, Mg2+, Pb2+, Ag+, Na+, Ca2+, Ba2+ and K+). As displayed in Fig. S3, probes 1 and 2 (20 μM in PBS buffer solution, pH = 7.54) towards the addition of 2 equiv. of various metal ions. 1 and 2 reveals the remarkable color changes from colorless to reddish brown and dark yellow colored with Cu2+. Whereas, no prominent color changes were observed with other metal ions. The colorimetric sensing studies are clearly indicates that, these probes can be utilized for the selective detection of Cu2+ ions. 3.3. Spectral response to metal ions The absorption and fluorescence manner response of the probes towards various metal ions were examined by UV-Vis and fluorescence spectroscopy correspondingly in PBS buffer solution (pH = 7.54). As illustrated in Fig. 3, absorption spectra of the probes 1 and 2 displayed the characteristic peak around 350 nm and 360 nm. The changes in photonic properties with the addition of metal ions like Cu2+, Fe2+, Co2+, Ni2+, Zn2+, Mn2+, Mg2+, Pb2+, Ag+, Na+, Ca2+, Ba2+ 11

and K+ as exhibited in Fig. 3a and b. In a typical experiment, upon addition of Cu2+ (0-2 equiv.) the probes 1 and 2 displayed the new intense band at 460 nm and 470 nm was gradually increased along with the increment of absorption band around 350 nm and 360 nm respectively (Fig. 3c and d). These results are obviously indicated that ligand to metal charge transfer (LMCT) between the probes and Cu2+ ions with the successive chelating complex formation attributes the remarkable color changes. There was no significant spectral changes were observed in the presence of other competing metal ions for both the probes (Fig. 3a and b), indicating that the UV-Vis spectral response of probes 1 and 2 is highly specific and selective detection of Cu2+. Based on UV-Vis titration data, the association constant [42] values of 1-Cu2+ and 2-Cu2+ was calculated to be 4.64 × 104 M-1 and 2.12 × 104 M-1 as depicted in Fig. S4. The Job’s plot analysis indicates the binding stoichiometry ratio of 1 and 2 with Cu2+ ion is 1:1 (Fig. 3e and f). The limit of detection of probes 1 and 2 for Cu2+ was found to be 0.303 μM and 0.345 μM as shown in Fig. S5, based on the equation LOD = K × SD/S, where K = 3, SD is the standard deviation of the blank solution, and S is the slope of the calibration curve [43]. (Figure 3) More interestingly, the high sensitivity of probes towards Cu2+ was executed by fluorescence spectroscopy (Fig. 4). The probes (1 and 2) reveals the intensive emission band at 645 nm and 656 nm was observed with the selective addition of Cu2+ ion. Whereas, the other tested metal ions did not display any noticeable spectral changes (Fig. 4a and b). Besides, the sensitivity of 1 and 2 with the incremental addition of Cu2+ (0-2 equiv.), the fluorescence spectra exhibits the intensities of the emission band at 645 nm for 1 and 656 nm for 2 was steadily increased with moderate blue shift (Fig. 4c and d). The enduring increase in emission band was caused by the interaction of Cu2+ with the probes 1 and 2 on the basis of the chelation enhanced 12

fluorescence effect (CHEF) [44]. The association constants (Ka) of Cu2+ with the probes can be obtained from the fluorimetric titration methods were calculated to be 1.51 × 105 M-1 for 1 and 7.74 × 104 M-1 for 2 (Fig. S6) respectively, by using Benesi-Hildebrand equation. Next, to find out the limits of detection was calculated by emission linear fitting for 1-Cu2+ and 2-Cu2+ as 0.22 μM and 0.729 μM (Fig. 5), which is compared with other reported values (Table 1). These limits are much lower than the limit of copper (∼20 μM) in drinking water by the U.S. Environmental Protection Agency [45], representative that it calculate Cu2+ in lower than micromole level. The LOD for Cu2+ detection with the probes 1 and 2 is sufficient to detect Cu2+ ion in living cells and biological systems. Finally, to resolve the dependence of fluorescence intensity (I/I0) with respect to [Cu2+]0/[R]0 for 1 and 2, where I and I0 are the emission intensities of the probes in the presence and absence of Cu2+, [Cu2+]0 and [R]0 are the concentration of Cu2+ and the probes. The fluorescence enhancement factor (FEF) was attained from (Fig. S7). FEF was found to be 7.7 for 1 and 3.7 for 2. These data was clearly indicated that the 1 has higher sensitivity with Cu2+ than 2. (Figure 4) (Figure 5) (Table 1) Moreover, the IR spectrum support for the interaction between 1-Cu2+ and 2-Cu2+was carried out in the KBr disc in the range of 4000-400 cm-1 (Fig. S8). The IR spectrum of the free probes (1 and 2) exhibited the broad band at 3411 and 3444 cm-1 due to the presence of Ph-OH group and this peak was disappeared upon chelating with Cu2+ ions, it suggests that the interaction between the phenolic OH with Cu2+. The νC=N peak at 1633 and 1615 cm-1 for 1 and 13

2, and this band was shifted to lower frequencies are caused by the interaction with Cu2+ ions at 1592 and 1602 cm-1. These results are obviously indicated the contribution of azomethine nitrogen in coordination with Cu2+ ion. The new band appeared at 513, 621 cm-1 due to ν(M-N) and 440, 459 cm-1 characteristics to ν(M-O) for 1-Cu2+ and 2-Cu2+, that the new peaks were not monitored in the free receptors. Then the further evidence for the formation of 1:1 stoichiometry of 2 with Cu2+ ion was recognized by ESI-MS spectrum (Fig. S9). The positive ion mass analysis reveals that the formation of 2 [Calcd: 339.839, m/z (M+H+): 340.35] and 2-Cu2+ [Calcd: 402.373, m/z (M+H+): 403.27]. From these spectrum results reveal that the 1:1 binding mode for probes and Cu2+ ion. 3.4. Competitive selectivity of 1 and 2 for Cu2+ with other metal ions The feasible interferences from other metal ions were further determined using interference experiments. Interferences of metal ions were resolved by adding 1 and 2 with 2 equiv. of Cu2+ in the presence of 3 equiv. of various competitive metal ions. As demonstrated in Fig. 6, compared with the probes 1 and 2 solutions in the presence of Cu2+ only, the absorbance and fluorescence intensities of 1 and 2 solutions which including both Cu2+ ion and the other metal ions exhibits no conspicuous difference. All these observations suggest that the probes 1 and 2 shows a highly superior binding of Cu2+ towards other metal ions. The combined results indicate the probes can be utilized to recognize Cu2+ ion without interference from other metal ions, and is a good fluorescent probes for detection of Cu2+. Consecutively to study the reversible and the reproducible nature of the probes with Cu2+, EDTA titrations were performed. As shown in Fig. S10, the naked-eye detection changes were clearly replicated in spectral measurements. The fluorescence of the 1-Cu2+ and 2-Cu2+ is quenched by the addition of EDTA as well as the UV-Vis spectra of the 1-Cu2+ and 2-Cu2+ band disappeared and recurrence of the original probes 14

band, Suggesting that Cu2+ has been removed from the probes and it specified the complete reversibility due to the interaction of EDTA with 1-Cu2+ and 2-Cu2+. These results are obviously indicates that the capability of probes and [1-Cu2+ and 2-Cu2+] ensemble for quantitative detection of Cu2+ and EDTA respectively as an “off-on-off” type probes. The possible mechanism for sensing Cu2+ and EDTA with probes 1 and 2 as depicted in Scheme 2. (Figure 6) (Scheme 2) 3.5. Molecular logic function The reversible and reproducible process of the probes 1 and 2 was also studied in a molecular logic circuit. Here, Cu2+ and EDTA as the two chemical input. The acquired absorbance and fluorescence spectroscopy data are given as chemical inputs ([Cu2+] and [EDTA]) and the obtained absorbance intensity at 460 nm (1) and 470 nm (2) as the output. Whereas, the fluorescence intensity at 645 nm (1) and 656 nm (2) as another output of the molecular logic circuit. These molecular logic circuits are combined with OR, AND, NOT and NOR gates. Based on the obtained spectroscopy data, we constructed the truth table. From the truth table, 1 and 2 were clearly mimic the given logic gate (Fig. 7a and b). The resultant absorbance and fluorescence spectral as presented in Fig. S10. (Figure 7) 3.6. Computational methods To demonstrate the mode of interaction between probes and Cu2+ ion, we have executed the density functional theory (DFT) calculations by means of B3LYP/6-31G (d,p) and B3LYP/LANL2DZ basis set using Gaussian 09 program [46, 47]. Fig. 8a and b are displayed the 15

attained higher electron density of the probes 1 and 2 on the imine moiety at HOMO and this electron cloud is transferred to the aldehyde moiety at LUMO. The appendage of Cu2+ ion to 1 and 2, electrons are occupied in the imine and metal part at HOMO and its spread out over the probe at LUMO. Moreover, the dipole moment of the probes 1 and 2 is also low compared to the Cu2+ complex. It reveals that the 1 and 2 are highly polarized by Cu2+ ions and displayed higher dipole moment. In addition, the energy gap between HOMO and LUMO is decreased by Cu2+ ions with 1 and 2. The optimized energy, dipole moment, RMS gradient Norm and HOMOLUMO energy gaps are listed in Table S3 and S4 for 1 and 2 respectively. (Figure 8) The sensing mechanism of 1 and 2 was further confirmed by time-depended density functional theory (TD-DFT) using aforementioned basis sets. The transitions of the 1 and 2 showed HOMO-LUMO (369 nm, 87.64% contribution and 393 nm, 95.15% contribution respectively) and HOMO-1 to LUMO (317 nm, 80.12% contribution and 328 nm, 60.03% contribution) are mainly contributing to the electronic configurations. Among them, HOMOLUMO transition is dominating than the HOMO-1 to LUMO transition, which is also agreed with the experimental wavelengths (350 nm and 360 nm, respectively). The copper complex of probes 1 and 2 predominately displays HOMO-1 to LUMO transition and it exhibits major oscillator strength at 412 nm and 424 nm respectively, due to LMCT (Table S5 and S6). From this analysis the Gibbs free energy can be calculated. The proposed probes 1 and 2 have lower energy than its copper complexes. These results are clearly indicating that the occurred strong interaction between the Cu2+ ions and with 1/2. The order of the energy is 1-Cu2+> 1 and 2Cu2+> 2. The change in enthalpy, energy, Gibbs free energy and entropy are listed out in Table S7 and S8 for 1 and 2 respectively. 16

3.7. Real sample analysis To examine the potential ability of the probes (1 and 2) for Cu2+ detection in three real samples like tap water, waste water and river water samples. Each experiment was conducted at least three times to obtain the concordant values and the attained results enhance the excellent recovery in real samples, which clearly demonstrate the probes was applicable for detection of Cu2+ in real samples. The observed results are illustrated in Table S9. 3.8. Application in living cells We evaluated the potential efficacy of probes 1 and 2 for fluorescence imaging of Cu2+ in living cell. The cytotoxicity of these probes to HeLa cells was evaluated by standard MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The results manifestly indicate that the probes are non-toxic to the HeLa cells under the experimental conditions. To observe the applicability of the probes in biological systems, it was applied to human cervical cancer cells (HeLa), here receptors and Cu2+ were endurable to uptake by the cells of attention and the images of the cells were taken by fluorescence microscopy with 325 nm excitation as displayed in Fig. 9 and 10. HeLa cells were incubated with 1 and 2 (5.0 µM, 1% DMSO) exhibits almost no fluorescence in the intracellular region after adding CuCl2 (5.0 µM) to the cells, they revealed strong intracellular fluorescence. These results evidently demonstrate the practical applicability of these probes for Cu2+ ions detection in living cells by fluorescence imaging. (Figure 9) (Figure 10) 4. Practical application 17

Some potential and practical applications of probes were previously reported [48, 49]. The successful color changes of probes with various metal ions were obtained in solution medium, the test kits were prepared by submerging filter paper into an in PBS buffer solution of 1 and 2 (0.05 M) and then drying for an hour in vacuum (oven temperature 50-60oC). The test kits containing 1 and 2 were applied to sense different metal ions (Fig. S11). After that, the different test kits were immersed in an aqueous solution of various metal ions for 1.0 min. An immediate color change was scrutinized only Cu2+ solution. Whereas, these test kits were concerned for recognizing towards different concentrations of Cu2+ (1×10-3 M and 1×10-4 M) reveal the different color change was obtained by naked-eyes. Hence, it is obviously exhibiting that distinct concentration of Cu2+can be as low 1×10-4 M. In order to investigate the another technique for detection of Cu2+ with the probes 1 and 2 also performed in the solid state. When the silica gel (60-120 mesh, 1.0g, Colorless) was preserved with 1 and 2 (buffer solution, 5 mL, 1×10-3 M) and solvent eradicated, colorless was imparted to silica (Fig. 11). When 1 and 2 were treated with 5 mL solution of Cu2+ at three different concentrations (0.01 M, 0.001 M, 0.0001 M), the intense color changes were observed from colorless to various colored respectively. After that, the solvent was removed under reduced pressure and the acquired colored silica powder was dried in an oven. The obvious color change was identified, the probes 1 and 2 can be utilized for practical applications. (Figure 11) 5. Conclusion In conclusion, we have designed and synthesized colorimetric probes (1 and 2) which provide as a selective and sensitive chemsensor that exclusively detect Cu2+ ion towards the

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other competing ions in PBS buffer solution using absorbance and fluorescence spectroscopy. Then, 1:1 binding stoichiometry was proposed based on a Job’s plot, ESI-MS and FT-IR analyses. Besides, Cu2+ binding capability with probes was further demonstrated using DFT calculations. To the best of our knowledge, the probes 1 and 2 has the possibility of detecting Cu2+ via colorimetric and fluorescent modes. Probes 1 and 2 enhanced the selective detection of Cu2+ at a low detection limit. Based on the detection limit, these probes were successfully applied to detect trace amounts of Cu2+ in real water samples. Moreover, these probes have been used for fluorescence imaging of Cu2+ in HeLa cells. Additionally, the test strips containing 1 and 2 have also exhibited a good selectivity and sensitivity of Cu2+ in solution medium and in addition to when supported on solid medium (silica). Acknowledgements We gratefully acknowledge the DST-SERB (Ref. No: SB/FT/CS-130/2012) for financial support. The authors are honestly expressed their heartfelt thanks to Sethu Institute of Technology, Kariapatti for their lab facilities and moreover thank to Madurai Kamaraj University and Mohamed Sathak Engineering College, Kilakarai for their instrumentation facilities. GS thanks Instem and DST-SERB for National post-doctoral fellowship (PDF/2016/000742).

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Author Biographies

Gujuluva Gangatharan Vinoth Kumar is currently doing Ph.D in the field of Colorimetric sensor at Chemistry Research Centre, Mohamed Sathak Engineering College, Kilakarai, Tamil Nadu, India. Mookkandi Palsamy Kesavan is currently doing Ph.D in the field of Nano drug delivery at Chemistry Research Centre, Mohamed Sathak Engineering College, Kilakarai, Tamil Nadu, India. Gandhi Sivaraman is currently working as SERB- National post-doctoral fellow in institute for stem cell biology and regenerative medicine, National centre for Biological sciences, Bangalore – 560065, India. His research interests are Chemical Biology, Fluorescence based sensors, Biomaterials, Drug delivery and Computational chemistry. Balasubramaniyam Sridhar is currently working in the of Laboratory of X-ray Crystallography, Indian Institute of Chemical Technology, Hyderabad, India. He is currently doing research in the field of X-ray crystallography. Kandasamy Anitha has received her Ph.D degree from School of Physics, Madurai Kamaraj University, Madurai, Tamilnadu, India. She is currently working in the field of X-ray crystallography. Jamespandi Annaraj has received his Ph.D degree from School of Chemistry, Madurai Kamaraj University, Madurai, Tamilnadu. He is currently working towards Nano biomaterials and Bioinorganic chemistry.

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Jegathalaprathaban Rajesh has received her Ph.D degree from School of Chemistry, Madurai Kamaraj University, Madurai, Tamilnadu, India. She is currently working towards Sensors, Nano drug delivery and Bioinorganic chemistry.

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Figures

Fig. 1. Molecular structure of the compound 2 with 50% probability thermal displacement ellipsoids. Fig. 2. (a) Packing diagram of the compound 2 viewed down along b-axis of the unit cell. Fig. 2. (b) Self associated ring S(6), S12(5) motifs, ring R22(14) motif and chain C22(7), C22(8) motifs extending along a-axis of the unit cell. Fig. 3. (a) & (b) Competitive absorption spectra of 1 and 2 (20 μM) in PBS buffer solution (pH = 7.54) containing 1% DMSO with 2 equiv. of different metal ions. (c) & (d) Absorption titration spectra of 1 and 2 (20 μM) with the sucessive addition of Cu2+ ion (0-2 equiv). (e) & (f) Job’s plot analysis of 1-Cu2+ and 2-Cu2+. The total concentration of 1, 2 and Cu2+ as 10 μM. Fig. 4. (a) & (b) Competitive fluorescence spectra of 1 and 2 (20 μM) in PBS buffer solution with 2 equiv. of different metal ions. (c) & (d) Fluorescence titration spectra of 1 and 2 (20 μM) with the incremental addition of Cu2+ ion (0-2 equiv.). λex = 325 nm for 1 and 2. Fig. 5. (a) Fluorescence spectra of the calibration curve of 1-Cu2+. (b) Fluorescence spectra of the calibration curve of 2-Cu2+. Fig. 6. (a) & (b) Competitive studies of 1 with Cu2+ (2 equiv.) and other metal ions (3 equiv.). (c) & (d) Competitive studies of 2 with Cu2+ (2 equiv.) and other metal ions (3 equiv.). Fig. 7. (a) Molecular logic circuit and its truth table of probe 1. (b) Molecular logic circuit and its truth table of probe 2.

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Fig. 8. Electron density maps of the frontier molecular orbital structures of (a) 1 and 1-Cu2+ (From left to right). (b) 2 and 2-Cu2+ (From left to right). Fig. 9. Bright field image and fluorescence image of HeLa cells (a) Bright field image HeLa cells incubated with probe 1 (5.0 μM) for 10 min at 37°C. (b) Fluorescence image of HeLa cells incubated with probe 1. (c) Bright field image of probe 1 treated HeLa cells again incubated with Cu2+ (5.0 μM). (d) Fluorescence image of probe 1 treated HeLa cells and with Cu2+. Fig. 10. Bright field image and fluorescence image of HeLa cells (a) Bright field image HeLa cells incubated with probe 2 (5.0 μM) for 10 min at 37°C. (b) Fluorescence image of HeLa cells incubated with probe 2. (c) Bright field image of probe 2 treated HeLa cells again incubated with Cu2+ (5.0 μM). (d) Fluorescence image of probe 2 treated HeLa cells and with Cu2+. Fig. 11. (a) Color changes of 1 (0.001 M) in the solid state upon addition of Cu2+ (from left to right: 1; 1+0.0001 M Cu2+; 1+0.001 M Cu2+; 1+0.01 M Cu2+). (b) Color changes of 2 (0.001 M) in the solid state upon addition of Cu2+ (from left to right: 2; 2+0.0001 M Cu2+; 2+0.001 M Cu2+; 2+0.01 M Cu2+).

Schemes Scheme 1. Synthetic protocol of Schiff base probes (1 and 2). Scheme 2. The plausible mechanism for sensing Cu2+ and EDTA with the probes 1 and 2.

30

Tables

Table 1. Detection limits of present work to compared with other reported values. Table 1 Probe

Method

Solvent system

Detection

References

limit Rhodamine-based

Rhodamine-based

Chromogenic and

Acetonitrile-

fluorescent

water

Colorimetric

Methanol-

4.1×10-5

[50]

1×10-6

[51]

HEPES Schiff base

Colorimetric

Acetonitrile

1.568×10-6

[3]

Graphene oxide

Electrochemical

Water

2.7×10-6

[52]

Rhodamine-based

Colorimetric and

DMSO/Tris-HCl

3.42×10-6

[53]

Fluorescent

buffer

Colorimetric /

DMSO/PBS

2.2×10-7 and

Present work

Fluorescence

buffer

7.29×10-7

Schiff base (1 and 2)

31

Fig. 1.

1

Fig. 2. (a)

2

Fig. 2. (b)

3

Fig. 3.

4

Fig. 4.

5

Fig. 5.

Fig. 6.

6

Fig. 7.

7

Fig. 8.

8

Fig. 9.

9

Fig. 10.

10

Fig. 11.

11

Scheme 1.

12

Scheme 2.

13