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Highly selective turn-on fluorescent sensor for nanomolar detection of biologically important Zn2 þ Based on isonicotinohydrazide derivative: Application in cellular imaging Kundan Tayade, Suban K Sahoo, Banashree Bondhopadhyay, Vimal K. Bhardwaj, Narinder Singh, Anupam Basu, Ratnamala Bendre, Anil Kuwar www.elsevier.com/locate/bios

PII: DOI: Reference:

S0956-5663(14)00394-7 http://dx.doi.org/10.1016/j.bios.2014.05.053 BIOS6815

To appear in:

Biosensors and Bioelectronics

Received date: 26 February 2014 Revised date: 3 May 2014 Accepted date: 5 May 2014 Cite this article as: Kundan Tayade, Suban K Sahoo, Banashree Bondhopadhyay, Vimal K. Bhardwaj, Narinder Singh, Anupam Basu, Ratnamala Bendre, Anil Kuwar, Highly selective turn-on fluorescent sensor for nanomolar detection of biologically important Zn2 þ Based on isonicotinohydrazide derivative: Application in cellular imaging, Biosensors and Bioelectronics, http: //dx.doi.org/10.1016/j.bios.2014.05.053 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 galley proof before it is published in its final citable 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.

Highly selective turn-on fluorescent sensor for nanomolar detection of biologically important Zn2+ based on isonicotinohydrazide derivative: Application in cellular imaging

Kundan Tayadea, Suban K Sahoob, Banashree Bondhopadhyayd, Vimal K. Bhardwajc, Narinder Singhc, Anupam Basud, Ratnamala Bendrea*, Anil Kuwara* a

School of Chemical Sciences, North Maharashtra University, Jalgaon-425 001 (MS), India

b

Department of Applied Chemistry, SV National Institute Technology, Surat (Gujrat), India.

c

Department of Chemistry, Indian Institute Technology, Ropar-140 001 (Punjab), India.

d

Molecular Biology and Human Genetics Laboratory, Department of Zoology, The

University of Burdwan, Burdwan (WB) India. *Corresponding

author

(Anil

Kuwar):

E-mail:

[email protected],

[email protected] Tel.: +91-257-2257432; Fax: +91-257-2257403.

Abstract A new Zn2+ selective chemosensor (3) was synthesized by condensation of commercially

available

substituted

salicylaldehyde

and

isonicotinohydrazide,

and

characterized by single crystal X-ray crystallography. Receptor 3 with Zn2+ exhibited a highly selective and pronounced enhancement in the fluorescence emission among different cations by forming a 2:1 complex. The receptor can detect Zn2+ up to nanomolar level (6.75 nM) with good tolerance of other metal ions and can be used for in vitro cellular imaging. Keywords: Fluorescent sensor, Zn2+ detection, Nanomolar detection, Cellular imaging

1. Introduction The expansion of fluorosensors for biologically active metal ions and their applications in the field of material, biological and environmental sciences are always significant, and therefore created a versatile research approaches and mechanisms for the designing and synthesis of novel probes containing more than one binding sites and signallingunits.1-3Along with the various transition metal ions, Zn2+ has attracted an immense arrangement of attention attributing to its biological importance. Zinc is the second most abundant and essential transition element in the human body behind iron, and known to involve in various biological processes such as catalytic centres and structural cofactors of many enzymes, DNA-binding proteins and neuronal signal transmission.4,5 Minute quantity of Zn2+ is required for the living organisms, but either deficiency or excessive amount can have various detrimental effects. Many severe neurological diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), hypoxia ischemia, Guam ALSParkinsonism-dementia Parkinson’s disease, infantile diarrhoea, and epilepsy are known to closely associate with the disorders of zinc metabolism.6-10 Also, the excessive concentration of zinc in the environment may reduce the soil microbial activity resulting in phytotoxic effect.11As a result, a responsive and undamaging technology to detect Zn2+ in environmental and biological systems, particularly in the occurrence of probable challenging cations like Cd2+, becomes very important. Because Zn2+ does not give any spectroscopicor magnetic signals due to its diamagnetic nature, the detection of Zn2+ in biological process cannot be measured by the common analytic techniques such as nuclear magnetic resonance (NMR), Mossbauer spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy. So the fluorescence spectroscopy is adopted as a superior selection for the actual point and genuine break recognition of Zn2+ in biological system without destructive them.

2-hyroxyl-3-isopropyl-6-methylbenzaldehydeis a popular substrate for nucleophilic addition reaction by virtue of its carbonyl group which can be activated intra-molecularly by the neighbouring phenolic hydroxyl group through hydrogen-bonding.12-15 Based on this concept, chemosensors with 2-hyroxyl-6-isopropyl-3-methyl benzaldehyde functionality as the binding site have been developed for the selective detection of different cations and anions. In this communication, we have developed a novel receptor 3 with a highly selective and sensitive fluorescent ‘turn-on’ response towards Zn2+ over the other surveyed cations including Cd2+. 2. Experimental All chemicals were purchased from Sigma–Aldrich.The absorbance and fluorescence spectra were recorded on a Shimadzu UV-Visible 2400 spectrophotometer and HORIBA JOBIN YVON Fluoromax-4 Spectrofluorometer, respectively.1HNMR spectra were recorded on a Bruker ARX 400 spectrometer using tetramethylsilane (TMS) as an internal standard. Elemental analyses were performed on a Perkin Elmer elemental analysis instrument.The infrared spectra were recorded on a Perkin Elmer Spectrum spectrometer using Nujol Mull. Mass spectra of receptor 3 and its complex with Zn2+ were obtained on a Bruker Ultraflex II MALDI/TOF spectrometer. 2.1 Synthesis of N'-(2-hydroxy-3-isopropyl-6-methylbenzylidene)isonicotinohydrazide(3) A solution of 2-hyroxyl-3-isopropyl-6-methyl benzaldehyde (0.001 M, 0.178 g) in anhydrous ethanol (25 ml) was added drop wise to a solution of isonicotic acid hydrazide (0.001 M, 0.137g) in anhydrous ethanol (25 mL). The mixture was refluxed for 5 hrs. Afterwards the content was kept at room temperature to obtain the precipitates, which were filtered and recrystallised from ethanol.Yield: 80 %, m.p. 2050C; IR (KBr, cm-1): υ = 1602, 1668, 3339; 1H NMR (CDCl3+DMSO-d6, δ ppm) 1.23 (d, 6H, gem 2 CH3), 2.40 (s, 3H, ArCH3), 3.37 (septet, 1H, CH), 6.68-717 (d, 2H, Ar-H, Ar-H), 7.76 (d, 4H, Ar-H, pyridine),

8.93 (s, 1H, CH = N), 11.96 (s, 1H, NH);CHN Analysis; Calcd. C, 68.67; H, 6.44; N, 14.13; Found C, 68.65; H, 6.66; N, 14.34. 2.2 UV–visible and fluorescence spectral measurements The metal ions Na+, K+, Ca2+, Mg2+, Al3+, Ba2+, Ce3+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Hg2+, Pb2+, Cd2+, Th4+ and Ag+ were added as their nitrate salts whereas Sr2+ and Mn2+ were added as chloride salts for absorption and fluorescence spectroscopic experiments. Stock solutions of metal ions (1×10-3 M) and the receptor 3 (1×10-4 M) were prepared in CH3OH. These stock solutions were used after appropriate dilution. Consecutively, to determine the stoichiometry of the receptor 3.Zn2+complex, the solutions of receptor 3 and Zn2+ were prepared at ratios of 3.0:0.0, 2.7:0.3, 2.4:0.6, 2.1:0.9, 1.8:1.2, 1.5:1.5, 1.2:1.8, 0.9:2.1, 0.6:2.4, 0.3:2.7 and 0.0:3.0. The fluorescence intensity of emission peak maxima at 554 nm was used for stoichiometry calculations. The plot of [HG] versus Xi was used to determine the stoichiometry of the complex formed. The concentration of [HG] was calculated by the equation of [HG] = (∆F/F0) [H] and Xi = Mole Fraction = [H]v/[H]v+ [G]v. 2.3 In vitro cell imaging with receptor3 and Zn2+ HeLa cells were procured from National Centre for Cell Sciences, Pune, India and grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1% L-glutamine-penicillin streptomycin. The cells were maintained at 37°C in a humidified atmosphere of 5% CO2. Cells after reaching 80-90% confluence were trypsinized and seeded in a 35mm culture dish with a seeding density of 3 × 105 cells per dish. The cells were treated with receptor 3 (0.98μM) for 30 min. At the end of 30 min, Zn2+ (10μM) were added and the cells were observed under inverted fluorescence microscope using a UV filter (Leica DMI6000B). The fluorescence images of cells were captured through an attached CCD camera using acquisition software.

2.4 In vitro cytotoxicity testing The cells were seeded in 24 well - culture plate with a seeding density of 10 × 104 cells per well. After overnight incubation, previous media was replaced by fresh serum free media and receptor 3 alone, receptor 3 along with Zn2+ and Zn2+ alone were added to the respective wells and incubated for 30min. To the wells, 50μl of [3-(4,5-dimethylthiazol-2-yl)2,5 diphenyltetrazolium bromide (MTT, 5 mgml-1 in PBS) was added and incubated for 3hrs. Culture media was removed and reduced formazon was dissolved by adding 500μl of DMSO for 15 min to develop a soluble purple color and measured the absorbance at 590nm (Shimazdu double beam spectrometer). 3. Results and discussion 3.1. Synthesis and spectroscopic characterization One step condensation of 2-hyroxyl-3-isopropyl-6-methyl benzaldehyde with isonicotic acid hydrazide gave receptor 3 (Figure 1). The receptor 3 was characterized by NMR, IR spectroscopy, elemental analysis and single crystal X-ray crystallography (Figure S1-S3). The crystallographic data of 3 and its unit-cell contents are presented in Table S1 and Figure S4, respectively. The CIF file for receptor 3 was deposited in the Cambridge Structure Database with CCDC No 980735. H2N O OH

1

+

NH N

Ethanol

N NH OH

O

N O

2

3

 

Figure 1.Synthesis of receptor 3

3.2. X-ray crystal structure studies Hydrogen bonding of the phenolic-OH was major advantageous feature in host-guest complexation.14 Invariability, phenolic hydrogen atom formed an intramolecular hydrogen bond to imine-N atom to give a six membered ring (Figure S3). This interaction was usually characterized in terms of phenolic oxygen to imine nitrogen separation.16

This distance

varies little between the two molecules. In all free receptor structure, the molecular involvement was via intramolecular hydrogen bonding.17

The receptor 3 exhibits

intramolecular hydrogen bonding (Table S2), where the H atom of phenolic hydroxyl group formed a strong O-H….N intramolecular hydrogen bond with O(1)….N(2) distance 2.56 Å which was in the middle of expected range of such hydrogen bonds. The longer bond distance of receptor 3 undergoes a solvent assisted keto tautomer leading to an isonicotamide hydrazine derivative of more chelating environment in presence of Zn2+ and exhibit high fluorescence intensity due to intramolecular charge transfer (ICT) process.18 3.3. Cation sensing The absorption spectral properties of receptor

3 were studied in CH3OH upon

addition of various metal ions such as K+, Ca2+, Mg2+, Al3+, Ba2+, Ce3+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Hg2+, Pb2+, Cd2+, Th4+ and Ag+. Free receptor 3 showed an absorption band at 308 nm. Upon addition of Zn2+, colour of the solution changed from colourless to yellow and the absorption band at 308 nm was red shifted to316 nm with the appearance of a new charge transfer band at 415 nm. Whereas in the presence of other metal ions (except for Cu2+), receptor 3 showed either no or slight change in the U.V. spectra (Figure S5). These results suggested a perturbation in the intramolecular charge transfer (ICT) character of the probe, due to the recognition of the Zn2+ at imine nitrogen, amide carbonyl and hydroxyl groups. This enhances the push pull character of the ICT state, and consequently a red-shift is observed upon deprotonation and charge transfers of the receptor by the cation. Further, upon

incremental addition of Zn2+ (0–2 equiv) to a solution of receptor 3, the intensity of absorption band at 308 nm (corresponding to pyridyl moiety) decreases and a new band appears at 415 nm with the formation of isosbestic points at 368, 347, 324 and 259 nm (Figure 2A). Receptor 20 µL 40 µL 60 µL 100 µL 140 µL 180 µL

Absorbance

1.5

1

10 µL 30 µL 50 µL 80 µL 120 µL 160 µL 200 µL

2.50E+06

Intensity (cps)

A 2

0.5

Receptor 20 µL 40 µL 60 µL 80 µL 100 µL 120 µL 140 µL 160 µL 180 µL 200 µL 300 µL

B

2.00E+06 1.50E+06 1.00E+06 5.00E+05 0.00E+00

0 210

260

310

360

410

460

Wavelength (nm)

415

515

615

Wavelength (nm)

Figure 2.(A) UV–vis absorption and (B) Fluorescence spectra (λex = 308 nm) of receptor 3 (0.1 mM) with increasing concentration of Zn2+ The fluorescence properties of 3 were further studied upon addition of 0.5 equiv. of different metal ions in CH3OH. The receptor 3 showed a weak fluorescence emission at 554 nm upon excitation at 308 nm. Interestingly, the fluorescence was remarkably enhanced in the presence of Zn2+ (Figure S6 and S7). However, there was no significant change in the emission behaviour of 3 with other metal ions. These results of fluorescence spectra can be explained with the facts that the azomethanine group of receptor 3 is poorly fluorescent due to the isomerization of the C=N double bond at the excited state alongwith theexcited-state proton transfer (ESPT) involving the phenolic proton of the substituted salicyldehyde moiety.19 Upon stable chelation with Zn2+, the C=N isomerization is inhibited. Also, the coordination of receptor 3 with the Zn2+ prevents the ESPT process, leading to the fluorescence enhancement. Moreover, the complexation reaction of Zn2+ with a chelating molecule induces rigidity in the resulting molecule and tends to produce a large chelationenhanced fluorescence (CHEF) effect whichcauses the large fluorescence enhancement.20

10 µL 30 µL 50 µL 70 µL 90 µL 110 µL 130 µL 150 µL 170 µL 190 µL 250 µL

To determine the specificity of 3, competitive experiments were carried out in the presence of Zn2+ (0.5 equiv.) mixed with other interfering metal ions (2 equiv.) As shown in Figure S8, no significant variation in the intensity was found with and without the other metal ions besides Zn2+. These results indicate that the receptor 3 shows a good sensitivity and selectivity towards Zn2+.The fluorescence titration of receptor 3 with Zn2+ ion resulted in a gradual enhancement in the fluorescence intensity (45 fold) at 554 nm (Figure 2B) with a detection limit of 6.75 nM, (Figure S9) which is comparable with reported values (Table 1).21-25 Table 1. Comparison of reported detection limit with present work Solvent System

Detection Limit

Response

References

DMF/H2O (7:3, v/v)

10 µM

Enhancement

21

DMSO/H2O (2:8, v/v)

15 µM

Enhancement

22

H 2O

0.2 µM

Ratiometric

23

DMF

11, 12 and 13 µM

Enhancement

24

Aqueous buffer

0.08 µM

Enhancement

25

CH3OH

6.75 nM

Enhancement

Present Work

The stoichiometry of the complex formed was determined with Job’s plot26 (Figure S10), and it was found to be 2:1. The proposition is further confirmed by the peak in the ESI-mass spectrum at m/z 612 corresponding to (3.Zn2+).The association constant Ka of 3 for zinc was calculated on the basis of Benesi-Hildebrand plot27 and found to be 1.00 × 104 M−1 (Figure S11). The recognition behaviour of the receptor 3 towards Zn2+ was further confirmed by FT-IR spectroscopy (Figure S12). The band corresponding to C=O observed at 1668 cm-1 was shifted to lower wave number indicating the participation of oxygen in the coordination.28 The absence of the bands at 1602 cm-1 in the metal complexes and their

negative shifts indicate the presence of enolic form of the receptor 3 and coordination through the nitrogen of C=N group. 3.4 Theoretical studies The density functional theory (DFT) method was employed to optimize the structure of the receptor 3 and its complex with Zn2+ by using the computer program Gaussian 09W.29 All the DFT calculations were carried out in the gas phase with a hybrid functional B3LYP (Becke’s three parameter hybrid functional using the LYP correlation functional) using the basis set 6-31G(d,p) for C, H, N and O atoms whereas LANL2DZ for Zn. The optimized structure of 3 and its complex with Zn2+ are shown in Figure S13. The root-mean-square error (RMSE) of 0.408 Å was obtained on superimposing the optimized and X-ray structures of 3. The computed bond parameters of 3 were found to be slightly higher than the X-ray results (Table S3) as these values were obtained for an isolated molecule in the gaseous phase.30 On complexation with Zn2+, there is an increase in the stability of the whole system which results in the decrease in the band gap between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) (Figure S13). Hence, the observed red-shift in the absorption spectra of 3.Zn2+can be explained in the terms of decreasedband gap of HOMO and LUMO. 3.5 In vitro cellular studies As shown in Figure 3, a significant fluorescence emission from the intracellular region was observed, suggesting a subcellular distribution of Zn2+in the cytoplasm. The cells after treatment with receptor 3 for 30 min showed very faint fluorescence under UV filter. When the cells were treated with Zn2+ along with receptor 3, the cells showed remarkable enhancement in fluorescence intensity (Figure 3E). Receptor 3 showed cytotoxic effect on the cells after 30 min treatment.

Figure 3 Live cell imaging with receptor 3 and Zn2+. (A) Phase contrast image of the HeLa without receptor 3 and Zn2+. B) The phase contrast image of the cells treated with receptor 3. C) The fluorescence image of the cells under UV filters after adding receptor 3. D) Phase contrast image of the cells treated with receptor 3 and Zn2+ E) Fluorescence image of the cells treated with receptor 3 and Zn2+ F) Fluorescence image of the cells treated with Zn2+ . The cells treated with both receptor 3 along with Zn2+ and only Zn2+ showed decrease in the viability. The cell viability sharply decreased in receptor 3 treated cells. To examine the utility of the sensor in biological systems, it was applied to human cervical cancer cell HeLa. Here, both the receptor 3 and Zn2+ were taken up by the cells of interest and the images of the cells were recorded by fluorescence microscopy under UV filter. There is a significant cytotoxic effect after 30 min. incubation with receptor 3 and Zn2+. This thus suggests that receptor 3 is slightly cytotoxic to the cells but can be utilized for short time fluorescence imaging of the cells (Figure 4).

Figure 4. The cells were treated with receptor 3 (0.98µM) and/or Zn2+(10µM) for 30min and followed MTT assay to measure the absorbance at 590nm. The viability of the cells readily decreased in receptor 3 treated cells with respect to the cells treated with receptor 3 along with Zn2+ and only Zn2+ compared to control and DMSO treated cells.

4. Conclusion A novel Zn2+ selective ‘turn-on’ fluorescent sensor 3 has been prepared which shows a 2:1 stoichiometry for metal ion. The receptor can exhibit significant changes in UV-vis, fluorescence, and mass spectra in the presence of Zn2+ with a detection limit of 6.75 nM. Morover, receptor 3 can be used for in vitro cellular imaging studies. References 1. Silva, A.P., de Gunaratne, H.Q.N., Gunnlaugsson, T., Huxley, A.J.M., McCoy, C.P. Rademacher, J.T., Rice, T.E., 1997. Chem. Rev. 97, 1515. 2. Valeur, B., Leray, I., 2000. Coord. Chem. Rev. 205, 3. 3. Zhang, J., Campbell, R.E., Ting, A.Y. Tisen, R.Y., 2002. Nat. Rev. Mol. Cell Biol. 3, 906. 4. Assaf, S.Y., Chung, S.H., 1984. Nature 308,734. 5. Scherz, H., Kirchhoff, E., 2006. J. Food Compost. Anal. 19, 420.

6. Frederickson, C.J., Koh, J.Y., Bush, A.I., 2005. Nat. Rev. Neurosci. 6, 449. 7. Fraker, P.J., King, L.E. Annu., 2004. Rev. Nutr. 24, 277. 8. Berg, J.M., Shi, Y., 1996. Science 271, 1081. 9. Koh, J.Y., Suh, S.W., Gwag, B.J., He, Y.Y., Hsu, C.Y., Choi, D.W., 1996. Science 272, 1013. 10. Bush, A.I., Pettingell, W.H., Multhaup, G., Paradias, M., Vonsattel, J.P., Gusella, J.F., Beyreuther, K., Masters, C.L., Tanzi, R.E., 1994. Science 265, 1464. 11. Voegelin, A., Poster, S., Scheinost, A.C., Marcus, M.A., Kretzschmar, R., 2005. Environ. Sci. Technol. 39, 6616. 12. Erdemir, S., Malkondu,S., 2013. Sensors and Actuators B 188, 1225. 13. Fegade, U., Sharma, H., Tayade, K., Attarde, S., Singh N., Kuwar, A., 2013. Org. Biomol. Chem. 11, 6824. 14. Butcher, R., Bendre, R., Kuwar, A., 2005. Acta Cryst. E61, o3511. 15. Kuwar, A., Shimpi, S. R., Mahulikar, P. P., Bendre, R. S., 2006. J. Sci. Ind. Res. 65, 665. 16. Kuwar, A., Fegade, U., Tayade, K., Patil, U., Puschmann, H., Gite, V., Dalal, D., Bendre, R., 2013. J. Fluoresc. 23, 859. 17. Butcher, R. J., Bendre, R. S., Kuwar, A. S., 2007. Acta Cryst. E63, o3330. 18. Kaur, K., Bhardwaj, V. K., Kaur, N., Singh, N., 2012. Inorg. Chem. Commun. 18, 79. 19. Hou, J.T., Liu, B.Y., Li, K., Yu, K.K., Wu, M.B., Yu, X.Q., 2013. Talanta 116,434. 20. Wang, J., Chu, Q., Liu, X., Wesdemiotis, C ., Pang, Y. J., 2013. Phys. Chem. B 117, 4127. 21. Kaur, K., Bhardwaj, V. K., Kaur N., Singh, N., 2013. Inorg. Chim. Acta. 399, 1. 22. Sivaraman, G., Anand, T., Chellappa, D., 2012. Analyst 137, 5881. 23. John, C.L., Huan, Y., Wu, X., Jin, Y., Pierce, D. T., Zhao, J. X., 2013. Analyst 138, 4950. 24. Ding, Y., Xie, Y., Li, X., Hill, J. P., Zhang, W., Zhu, W., 2011. Chem. Commun. 47, 5431. 25. Li, N., Xiang, Y., Chen, X., Tong, A., 2009. Talanta 79, 327.

26. Job P., 1928. Ann Chim. Appl. 9, 113. 27. Benesi, H.A., Hildebrand, J.H.,1949. J. Am. Chem. Soc. 71, 2703. 28. Narang, K.K., Aggarwal, A.A., 1975. Indian J.Chem.13, 1072. 29. Frisch, M.J. et. al. Gaussian 09, G09W®, Gaussian Inc., Wallingford, USA, 2009. 30. Sahoo, S.K., Sharma, D., Bera, R.K., 2012. J. Mol. Model. 18, 1993. Table 1. Comparison of reported detection limit with present work Solvent System

Detection Limit

Response

References

DMF/H2O (7:3, v/v)

10 µM

Enhancement

21

DMSO/H2O (2:8, v/v)

15 µM

Enhancement

22

H 2O

0.2 µM

Ratiometric

23

DMF

11, 12 and 13 µM

Enhancement

24

Aqueous buffer

0.08 µM

Enhancement

25

CH3OH

6.75 nM

Enhancement

Present Work

 

Highlight ¾ Zinc chemosensor was constructed through the selective assembly of a chemosensor. ¾ Fluorescence spectra, a DFT & IR spectrum clearly explains the binding mode between the chemosensor & the detected Zn2+. ¾ The developed sensor was successfully applied to image intracellular Zn2+ in living cells.