Accepted Manuscript Synthesis and characterization of zinc sulfide quantum dots and their interaction with snake gourd (Trichosanthes anguina) seed lectin Khan Behlol Ayaz Ahmed, Pichaikkannu Ahalya, S. Megarajan, Ravikanth Kamlekar, Veerappan Anbazhagan PII: DOI: Reference:

S1386-1425(15)30084-6 http://dx.doi.org/10.1016/j.saa.2015.07.035 SAA 13934

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

25 February 2015 5 June 2015 7 July 2015

Please cite this article as: K.B.A. Ahmed, P. Ahalya, S. Megarajan, R. Kamlekar, V. Anbazhagan, Synthesis and characterization of zinc sulfide quantum dots and their interaction with snake gourd (Trichosanthes anguina) seed lectin, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/ 10.1016/j.saa.2015.07.035

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Synthesis and characterization of zinc sulfide quantum dots and their interaction with snake gourd (Trichosanthes anguina) seed lectin Khan Behlol Ayaz Ahmeda, Pichaikkannu Ahalyaa, S. Megarajana, Ravikanth Kamlekarb and Veerappan Anbazhagana* a

Department of Chemistry, School of Chemical and Biotechnology, SASTRA University, Thanjavur, Tamil Nadu, India.

b

Environmental and Analytical Chemistry Division, School of Advance Sciences, Vellore Institute of Technology, Vellore - 632014, Tamil Nadu, India

*Corresponding author, V. A: Phone: +91 4362-264101-3689; Fax: +91 4362-264120, e-mail: [email protected]

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Abstract Owing to the use of quantum dots in biological labeling, and the specific interaction of lectins with tumor cells, studies on lectin-QDs interaction are of potential interest. Herein, we report a facile method to prepare zinc sulfide quantum dots (ZnS QDs) using pectin as a capping agent, and studied their interaction with snake gourd seed lectin (SGSL) by fluorescence spectroscopy. The QDs were characterized by X-ray diffraction, high-resolution transmission electron microscopy, UV-Vis absorption and fluorescence spectroscopy. The thermodynamic forces governing the interaction between ZnS-QDs and SGSL have been delineated from the temperature dependent association constant. These results suggest that the binding between ZnS QDs and SGSL is governed by enthalpic forces with negative entropic contribution. The red shift of synchronous fluorescence spectra showed that the microenvironment around the tryptophan residues of SGSL was perturbed by ZnS-QDs. The obtained results suggest that the development of optical bioimaging agents by using the conjugated lectin-QDs would be possible to diagnose cancerous tissues.

Key words: Zinc sulfide quantum dots; Pectin; Lectin; Snake gourd seed lectin; Binding; Thermodynamics

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1. Introduction Semiconductor quantum dots (QDs) has attracted researchers for their interesting optical and electronic properties [1-3]. These materials display potential application in photocatalysis, photovoltaics, electrochemical devices and microelectronics [4, 5]. QDs emerged as a better fluorescent probe than the traditional organic fluorophores used for biological labeling, because of their excellent photoluminescence, greater photostability, continuous excitation spectrum, size-tunable and narrow emission bands [6, 7]. In general, for the preparation of QDs, stabilizing agents such as trioctyl phosphine/trioctyl phosphine oxide, thiols, thioacids and amides was used. The most challenging part of using QDs in biological environment is their toxicity that arises due to the decomposition and release of the metal ions or that from the capping agents [8]. Derfus et al, have reported the degradation of CdSe QDs under UV irradiation which led to the release of cadmium ions and induce cell death [9]. Clift et al., showed that the different surface coated QDs can cause oxidative stress and affect cell signaling [10]. Therefore, it is imperative to prepare water-soluble, non-toxic and biocompatible QDs. Of late, QDs doped with transition metal ion have been explored to reduce the toxicity of QDs. Geszke-Moritz et al., have reported the preparation of folic acid conjugated thioglycerol capped Mn:ZnS dopped dots for imaging T47D breast cancer cells [11]. Santra et al., have reported manganese-doped Mn:CdS-ZnS core-shell QDs for labeling brain blood vessels [12]. ZnS is considered to one of the less toxic material with excellent photoluminescence properties [13]. The preparation of ZnS QDs involving toxic organic solvents was not suitable for biomedical application. The viable method to synthesize biocompatible ZnS QDs is to use non toxic chemicals and environmental benign solvents. In order to improve its practical applications, researchers started to modify the large surface area of QDs with biorecognition molecules, such 3

as proteins, peptides and antibodies [14,15]. Recently, Huang et al. reported that the binding between QDs and proteins are significantly influenced by the capping agents [16]. In this paper, we report a convenient method to synthesize stable zinc sulphide (ZnS) QDs using the biomolecule, pectin as a templating agent and taurine as an antioxidant and studied their interactions with lectin.

Taurine and pectin were chosen because it is benign and more

importantly, taurine plays a significant role in protecting the cells against oxidative damage [17]. Pectin is a biopolymer that consists of a linear backbone of (1-4) linked α-D-galacturonic acid residue with a varying degree of methylesterifed carboxyl group. Pectin is widely used as gelling, thickening and stabilizing agents in the food industry. The mucoadhesive natures of pectin have been exploited for in vivo delivery [18]. Lectins are widely known as carbohydrate binding proteins, but it can bind to other ligands too. For example, the polymers, porphyrin, adenine, 1,8-anilinonaphthalenesulfonate, 2,6toluidinylnaphthalenesulfonate, cytokinin etc., can bind to lectins with considerable affinity [19, 20]. Several lectins are known to agglutinate tumor cells; therefore conjugating QDs to lectins is expected to increase the selectivity of the conjugate to the tumor cells [21, 22]. Therefore, investigations on the interaction between QDs and lectins are important from a therapeutic perspective, in addition to their usefulness in learning lectin-QDs interactions. Snake gourd seed lectin (SGSL) is a heterodimeric glycoprotein with one or more intersubunit disulfide bridges and an apparent molecular weight of 60 kDa. It shows a high preference for β anomeric form of galactose over the corresponding α anomeric form. Chemical modification studies have shown that the histidine residues of SGSL are involved in the sugarbinding and hemagglutination activity [23-25]. Spectrofluorimetric and chemical modification studies have shown that the tryptophan residues of the SGSL are in the heterogeneous 4

hydrophobic environment [26]. SGSL binds strongly with carbohydrates derivatives bearing a hydrophobic group over other simple saccharides, indicating the existence of some hydrophobic site near the sugar-binding site [23-25]. These properties have been previously investigated to prepare SGSL-porphyrin conjugate to find application in photodynamic therapy [27]. Therefore, it is possible for SGSL to bind highly luminescent QDs, which may find application in biological labeling. To delineate the thermodynamic forces that govern the interaction, we performed binding experiments at different temperature. The results showed that SGSL-ZnS-QDs interaction was governed by enthalpic forces with negative entropy contributions. Additionally, synchronous fluorescence studies reveal that the interaction of ZnS-QDs induces a modification in the SGSL tertiary structure and produces perturbation around the microenvironments of the Trp residues of SGSL.

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2. Materials and Methods 2.1. Materials Analytical grade zinc chloride, sodium sulphide and Citrus pectin were purchased from Alfa Aesar, India and were used without further purification. Taurine was obtained from Sigma, India. The snake gourd seeds were obtained from local seed vendor. Millipore water was used for the preparation of all aqueous solutions. All the glass wares were cleaned with freshly prepared aqua regia (3:1, HCl: HNO3) and rinsed thoroughly with water. Then, they were dry sterilized in hot air oven at 160 oC for 3 h, prior to use. 2.2. Synthesis and characterization of QDs ZnS were synthesized by wet chemical method using analar grade sodium sulphide, Na2S.9H2O as a source for S2- ions and zinc chloride as the source for Zn2+ ions, with pectin as the capping agent and taurine as the antioxidant. Briefly, 50 mg of pectin, 1 mM of zinc chloride and 10 mM of taurine was dissolved in 50 mL of Millipore water and was refluxed for 30 min. To this reaction mixture, freshly prepared Na2S was injected (1mM final concentration) under vigorous stirring and refluxed for 8 h. The formation of QDs was confirmed by illuminating the reaction mixture under a UV lamp (inset of Fig. 1). The concentration of taurine plays an important role in synthesizing highly luminescent QDs. In fact, the synthesis performed in absence of taurine resulted in bigger aggregates with poor optical property. The synthesis performed only with taurine as a capping did not produce fluorescent QDs, suggesting that pectin is required for capping and taurine is required to prevent oxidation and agglomeration. The main advantage of this method is that the synthesis can be carried out in the aqueous environment in open air without the need for inert atmosphere. The synthesized QDs were characterized by X-

6

ray diffraction (XRD), high resolution transmission electron microscopy (HR-TEM), UV-Visible spectroscopy and fluorescence spectroscopy. The transmission electron micrographs of the synthesized QDs were measured on JEM 1011, JEOL, Japan. A drop of QDs was placed on a carbon-coated copper grid and was dried prior to the measurement. XRD measurements of the QDs were done on a XRD-Bruker D8 Advance X-ray diffractometer using monochromatic Cu Kα radiation. The optical absorbance of the synthesized QDs was monitored by UV-Vis spectrophotometer (Thermo Scientific Evolution 201) between wavelengths of 200 to 800 nm at a resolution of 1 nm. Fluorescence measurements were carried out on a JASCO spectrofluorometer FP 8200 with a 1.0 cm quartz cell. The fluorescence of QDs was recorded in 370 – 600 nm at an excitation wavelength of 360 nm and the slit widths of both excitation and emission were set at 5 nm. 2.3. Purification of snake gourd seed lectin Lectin (SGSL) form the seeds of Trichosanthes anguina were purified by an affinity chromatography on cross linked guar gum [28] as described previously [23-25]. The protein purity was judged by polyacrylamide gel electrophoresis. The protein concentration was determined by Lowry assay [29]. Bovine serum albumin was used as the standard. 2.4. Binding of QDs to SGSL The intrinsic fluorescence spectrum of SGSL was recorded in 300 – 400 nm range at an excitation wavelength of 280 nm. A fixed volume of SGSL solution (3.0 ml, 2.5 µM) was titrated by adding small aliquots of the QDs from a concentrated stock solution (1 mM) and the fluorescence intensity was measured after an equilibration period of 2 min. All measurements were performed at 293, 298, 303, 308, and 313 K. All binding experiments were performed in 10 mM phosphate buffer, containing 0.15 M NaCl, pH 7.4 (PBS). All the binding experiments were 7

repeated at least three times to arrive at the average values. Fluorescence intensities were corrected for volume changes and for inner filter effects before further analysis. The synchronous fluorescence spectrum was monitored by scanning simultaneously the excitation and emission wavelength at 80 or 30 nm intervals. 2.5. Lectin activity assay The activity of the protein was checked by haemagglutination and haemagglutination inhibition assays as described in ref. [23-25]. To evaluate whether the binding of QDs has altered the carbohydrate-binding activity of lectin, the haemagglutination experiments were performed by incubating lectin with high concentrations of QDs as employed in the fluorescence studies.

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3. Result and Discussion Zinc sulfide quantum dots (ZnS-QDs) were synthesized by using the biomolecules, pectin and taurine. Fig. 1 shows the UV-Vis spectra and fluorescence emission spectra of the freshly prepared ZnS-QDs. An absorption onset edge for ZnS-QDs was observed at 360 nm (Fig. 1). The band gap computed from the absorption edge for ZnS was 3.5 eV. The observed increases of the band gap as compared to bulk were due to the size effect, which are consistent with the previous report on semiconducting ZnS quantum dots [30, 31]. The fluorescence emission maximum of the ZnS-QDs was at 449 nm which was the typical luminescence spectrum related to defects in the ZnS [30, 31]. The XRD patterns of the synthesized ZnS-QDs are shown in Fig. 2. The characteristic diffraction peaks for zinc blend structure of ZnS-QDs was obtained at the 2θ values of 28.67o, 48.82o and 56.56o, corresponding to diffraction at (1 1 1), (2 2 0) and (3 1 1) planes, respectively (Fig 2) [31]. The peak broadening in the XRD patterns clearly suggests the formation of ZnS nanocrystal of small size. Fig. 3 shows the typical TEM image of the synthesized pectin capped ZnS-QDs. The average core size of prepared ZnS-QDs calculated from the statistical results was about 3.8 ± 0.4 nm (Fig. 3A and 3B). The appearance of fringe pattern in HR-TEM image and selected area electron diffraction (SAED) pattern confirm the formation of nanocrystalline QDs. The observed three rings in SAED can be indexed to (1 1 1), (2 2 0) and (3 1 1) lattice plane of cubic ZnS [31, 32]. Zeta potential estimated for ZnS QDs was -20.6 mV, indicating that QDs are stable due to electrostatic repulsion (Fig. S1). Lectins have been recognized as bioadhesive drug delivery systems [33-35]. Recently, Jacalin-phthalocyanine-gold nanoparticles conjugate were used to target HT-29 human colorectal adenocarcinoma cells [36].

As a first step towards developing lectin conjugated QDs for 9

targeting specific cells, we investigated the details of thermodynamic forces involved in the interaction between the QDs and SGSL. The potential interaction between SGSL and QDs was measured by monitoring the intrinsic fluorescence emission peak of SGSL at 334 nm. The results demonstrate that the fluorescence emission spectrum of SGSL decreases drastically upon increasing the concentration of QDs (Fig. 4A). Additionally, increasing the concentration of ZnS QDs resulted in a red-shift in the emission maximum, indicating that under the given experimental conditions tertiary structural changes are involved in QDs binding to SGSL. Furthermore, ZnS QDs binding to SGSL does not affect the far-UV circular dichorism spectra, indicating no substantial changes in the lectin secondary structure upon QDs binding (Fig. S2). As noted from Fig. 4A, the fluorescence intensity of SGSL in the presence of QDs was quenched with an increase in the fluorescence at 450 nm. This indicates that there is an effective energy transfer between SGSL and ZnS QDs, and the complex compound has been formed between QDs and SGSL. The formation of a complex between the lectin and ZnS-QDs was further evidenced by the appearance of an isosbestic point at 390 nm (Fig. 4). Notably, the addition of pectin and taurine to SGSL did not affect the SGSL fluorescence intensity, indicating that the observed quenching is due to the binding between ZnS QDs and SGSL (Fig. S3). Time resolved fluorescence of native SGSL showed two different lifetimes for the excited tryptophan residues, τ1 (1.53 ns) and τ2 (4.90), which is consistent with the previous report [26]. The addition of ZnS QDs to SGSL significantly decreases the fluorescence life times, τ1 (1.26 ns) and τ2 (4.29), indicating that one or more easily accessible tryptophan could be quenched by ZnS QDs (Fig. S4). Analyzing the fluorescence quenching data using Stern-Volmer equation shows a linear dependence of protein fluorescence with respect to QDs concentration (Fig. 4B), supporting the 10

complex formation between the SGSL-QDs [37, 38]. Zeta potential estimated for ZnS QDs upon binding to SGSL increases considerably from -20.6 mV to -27.9 (Fig. S5), indicating the higher stability of SGSL-ZnS complex. Moreover, the hydrodynamic size of ZnS QDs increases from 87.6 nm to 187.7 nm suggesting that the ZnS QDs is surrounded by the protein molecules (Fig. S6). Fig. 5A shows the typical binding curve for the association of ZnS-QDs with SGSL. The binding curve shows that the change in the fluorescence intensity increases with increasing ligand concentration, displaying the saturation behavior. The association constant (Ka) and the number of binding sites (n) between SGSL and ZnS QDs can be obtained by the following equation [38-40]. log (F0 - Fc/Fc - F∞) = log Ka + nlog[QDs]

(1)

where F∞ is the change in fluorescence intensity at infinite drug concentration. F∞ was obtained from the ordinate intercept of the plot of 1/∆F∞ vs 1/[QDs] (inset of Fig. 5B). A double logarithmic plot for the interaction of ZnS-QDs with SGSL is given in Fig. 5B. Using equ.1, n and K were determined at different temperature and are presented in Table 1. The values of n at the experimental temperatures were approximately equal to 1 (Table 1), indicating one class of the binding site to QDs in SGSL. In order to describe the thermodynamic force governs the interaction between SGSL and QDS, binding experiments were performed at different temperatures. The thermodynamic parameters, enthalpy of binding (∆H0) and entropy of binding (∆S0) associated with the interaction of ZnS were obtained by van’t Hoff plots (Fig. 6): lnKaT = (-∆H0/RT) + (∆S0/R)

(2)

where KaT is the association constant at the corresponding temperature, R is the gas constant. The van’t Hoff plot shows a linear relationship between lnKaT versus 1/T. The enthalpy and 11

entropy of binding for ZnS-QDs were obtained as ∆H0 = - 51.97 kJ mol-1 and ∆S0 = - 40.15 J mol-1 K-1. The enthalpy change and entropy change obtained from the plot (Fig. 6) yield the Gibbs free energy change (∆G0). ∆G0 = ∆H0 - T ∆S0

(3)

For the interaction of SGSL with ZnS-QDs, the ∆G0 calculated using eq. 3 at 25 oC, was obtained as - 24.61 kJ mol-1 The negative valve of ∆G0 suggests that the interaction of SGSL to the surface of ZnS-QDs is a spontaneous process. The thermodynamic parameters ∆H0 and ∆S0 obtained from the van’t Hoff plot of the KaT values for ZnS (Table 1) indicate that the binding of QDs is governed by enthalphic forces and that the contribution from entropy to the binding process is negative [37, 38]. Similar enthalpic favorable interactions were observed between cadmium telluride QDs and chymotrpysin [41] and cadmium sulfide QDs and hemoglobin [39]. The negative value of ∆H0 also indicates possible electrostatic interactions, as observed in the bovine serum albumin-QDs complex [42]. The negative value of ∆S0 is the common characteristics of hydrogen bonds and Vander Waals interaction [42, 43]. Based on the previous studies [42, 43], we propose that the pectin capped ZnS QDs association with SGSL must have a major contribution from polar interactions such as hydrogen bonding, as observed in the Momordica charantia lectin-porphyrin complex and Concovalin A-porphyrin complex [44, 45]. Moreover, any conformational changes of the lectin are expected to result in entropy gain, which might be one of the significant driving forces for SGSL binding to the surface of ZnS-QDs. The possible conformation changes of SGSL are investigated further by synchronous fluorescence. Synchronous fluorescence spectroscopies have been widely used to separate the overlapped excitation peaks of aromatic residues in conventional fluorescence spectrum [46-48]. 12

The synchronous fluorescence spectra of SGSL contributing to Tyr and Trp residues was obtained as described in methods. The peak at 313 nm is attributed to the Tyr residues, and the peak at 353 nm is attributed to Trp residues (Figure 7). The polarity of the environment has been known to influence the fluorescence emission peak of Tyr and Trp residues [46-48]. No shift in Fig. 7A suggests that the environment of Tyr residues in SGSL did not change in the presence of ZnS-QDs. The red shift observed in Fig. 7B suggests that the environment of Trp residues is altered considerably and moved to a hydrophilic environment due to the interaction between the SGSL and ZnS-QDs. These results clearly indicate that the binding of ZnS-QDs alters the tertiary structure of the adsorbed SGSL, and modify the microenvironments around the Trp residues and causes fluorescence quenching. Hemagglutination and the hemagglutination inhibition activity of SGSL in the absence and the presence of specific sugar, lactose is shown in Fig. S8. Agglutination-inhibition assay performed in the presence of ZnS-QDs or capping agent, pectin and taurine displays no loss of erythrocyte-agglutination activity by SGSL (Fig. S8B) [49]. However, the addition of 0.1 M lactose to SGSL-QDs inhibits agglutination indicating that the QDs and sugar binds at different sites on the lectin surface (Fig. S8B). These results further support that the SGSL remain in the functional form, while interacting with the QDs. The association constants obtained here for the SGSL-QDs complexes are in the range of 1.3 × 104 to 3.8 × 104 M-1 (Table 1). These values are comparable to those observed generally for lectin-monosaccharide complexes [50] as well as those obtained from lectin-porphyrin complexes. The binding constants of SGSL-QDs complexes were also compared with those that bind lectin with hydrophobic ligands and plant growth hormones, such as 2,6toluidinylnapthalene-sulphonic acid, adenine, auxins and cytokinins (Ka ~ 1.0 × 103 to 1.0 × 106 13

M-1) [19, 20]. The strength of the SGSL-QDs interactions also indicates the existence of other endogenous hydrophobic ligands for this lectin [51-55]. Since several lectins are known for selective agglutination of tumor cell, lectins can be proposed as carriers for the targeted delivery of drugs and pharmaceuticals to tumor tissues. Several lectin-drug conjugates have been reported and some of them display good efficacy when tested on cultured cells and animal models [56, 57]. In light of these observations and the considerable strong interaction between SGSL and QDs, it is possible to use lectin as a vehicle for targeting highly fluorescent QDs to specific tissues and also for biological labeling. Further, a simple affinity chromatography method to purify SGSL in relatively large quantities renders the SGSL-QDs conjugate for a wide range of application.

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4. Conclusion In this study, a facile method to synthesize water-soluble ZnS-QDs with pectin as capping agent has been described. This method is simple, eco-friendly and cost effective for producing QDs on a large scale. The proposed method can be extended to the preparation of other semiconductor quantum dots. The interaction of QDs with lectin has been explored for the first time. The fluorescence data revealed that the ZnS QDs effectively quenched the intrinsic fluorescence of SGSL via static quenching and affected the microenvironment of Trp. Thermodynamic parameters associated with the SGSL-QDs interaction reveals that QDs binding is a spontaneous process, in which enthalpic forces played a major role with a negative contribution from binding entropy. The reported high association constant of SGSL with ZnS-QDs, as good as specific sugars, indicates the potential of SGSL as a carrier for targeting these QDs to tumorous tissues. Additional studies with cell cultures and/or animal models would help in recommending these lectin–QD conjugates for biological and biomedical applications.

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Acknowledgements KBAA earnestly acknowledges the teaching assistantship from SASTRA University. The Department of Science and Technology, Government of India (SB/FT/LS-217/2012) is acknowledged for the financial support. We thank central research facility (R&M/0021/SCBT007/2012-13), SASTRA University for the infrastructure. We thank Dr. Venkatesan and Mr. Ganavel, Pondicherry University for their help in time resolved fluorescence.

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References 1. M. Ackermann, M.A. Perusal, S. Kos, D.L. Smith, D.D. Kopeks, V.I. Limo, Nature 429 (2004) 642-646. 2. X. Peng, L. Manna, W. Yang, J. Wickham, E. Scher, A. Kadavanich, A.P. Alivisatos, Nature 404 (2000) 59-61. 3. A.P. Alivisatos, Science 271 (1996) 933-937. 4. G.S. Li, D.Q. Zhang, J.C. Yu, Environ. Sci. Techno 43 (2009) 7079-7085. 5. Y. Yu, X. Chen, Y. Wei, J.H. Liu, S.H. Yu, X.J. Huang, Small 8 (2012) 3274-3281. 6. X. Michalet, F.F. Pinaud, L.A. Bentolila, J.M. Tsay, S. Dosse, J.J. Li, G. Sundaresan, A.M. Wu, S.S Gambhir, S. Weiss, Science 307 (2005) 538-544. 7. S.B. Rizvi, S. Ghaderi, M. Keshtgar, A.M. Seifalian, A.M, Nano Rev 1 (2010) 5161. 8. K.T. Yong, W.C. Law, R. Hu, L. Ye, L. Liu, M.T. Swihart, P.N. Prasad, Chem. Soc. Rev 42 (2013) 1236-1250. 9. A.M. Derfus, W.C.W. Chan, S.N. Bhatia, Nano Lett. 4 (2004) 11–18. 10. M. J. D. Clift, M. S. P. Boyles, D. M. Brown, V. Stone, Nanotoxicology 4 (2010) 139– 149. 11. M. Geszke-Moritz, H. Piotrowska, M. Murias, L. Balan, M. Moritz, J. Lulekb, R. Schneider, J. Mater. Chem. B 1 (2013) 698–706 12. S. Santra, H. Yang, J. T. Stanley, P. H. Holloway, B. M. Moudgil, G. Walter, R. A. Mericle, Chem. Commun. (2005) 3144–3146. 13. H. Li, W. Y. Shih, W-H. Shih, Nanotechnology 18 (2007) 205604. 14. L. Shao, Y. Gao, F. Yan, Sensors 11 (2011) 11736-11751. 15. W.C.W. Chan, D.J. Maxwell, X. Gao, R.E. Bailey, M. Han, S. Nie, Curr. Opin. Biotechnol 13 (2002) 40-46. 16. D. Huang, F. Geng, Y. Liu, X. Wang, J. Jiao, L. Yu, Colloids and Surfaces A: Physicochem. Eng. Aspects 392 (2011) 191-197. 17. S.A. Messina, R.Jr. Dawson, Adv. Exp. Med. Biol 483 (2000) 355-367. 18. P. Sriamornsak, Expert Opin. Drug Deliv 8 (2011) 1009-1023. 19. H. Lis, N. Sharon, Chem. Rev 98 (1998) 637-674. 20. S.S Komath, M. Kavitha, M.J. Swamy, Org. Biomol. Chem 4 (2006) 973-988. 21. E.G. De Mejía, V.I. Prisecaru, Crit. Rev. Food Sci. Nutr 45 (2005) 425. 22. L. Ding, P.J. Zhou, S.Q. Li, G.Y. Shi, T. Zhong, M. Wu, J. Fluoresc 21 (2011) 17–24. 23. 17. S.S. Komath, S.K. Nadimpalli, M.J. Swamy, Biochem. Mol. Biol. Int 39 (1996) 243252. 24. S.S. Komath, S.K. Nadimpalli, M.J. Swamy, Biochem. Mol. Biol. Int 44 (1998) 107-116. 17

25. S.S. Komath, M.J. Swamy, Current Sci 75 (1998) 608-611. 26. S.S. Komath, M.J. Swamy, J. Photochem. Photobiol. B 50 (1999) 108. 27. S.S. Komath, R. Kenoth, L. Giribabu, B.G. Maiya, M.J. Swamy, J. Photochem. Photobiol B 55 (2000) 49-55. 28. P.S. Appukuttan, A. Surolia, B.K. Bachhawat, Indian J Biochem Biophys 14 (1977) 382– 384. 29. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem 193 (1951) 265275. 30. J. Rahdar, Nanostruc. Chem 3 (2013) 10. 31. B. Barman, K.C. Sarma, Chalcogenide Lett 8 (2011) 171-176. 32. S. Liu, H. Zhang, M.T. Swihart, Nanotechnology 20 (2009) 235603 33. M. Gavrovic-Jankulovic, R. Prodanovic, J. Biomaterials and Nanobiotech. 2 (2011) 614621. 34. L. Neutsch, V.E. Plattner, S. Polster-Wildhofen, A. Zidar, A. Chott, G. Borchard, O. Zechner, F. Gabor, M. Wirth, J. Urol 186 (2011) 1481-1488. 35. X.Y. Wang, R. Koller, M. Wirth, F. Gabor, Int. J. Pharm 436 (2012) 738 - 743. 36. G. Obaid, I. Chambrier, M.J. Cook, D.A. Russell, Angew. Chem. Int. Ed. Engl 51 (2012) 6158-6162. 37. A. Veerappan, T. Eichhorn, M. Zeino, T. Efferth, D. Schneider, Phytomedicine 20 (2013) 969-974. 38. B.K. Paul, D. Ray, Guchhait. Phys. Chem. Chem. Phys 15 (2013) 1275-1287. 39. X.C. Shen, X.Y. Liou, L.P. Ye, H. Liang, Z.Y. Wang, J Colloid Interface Sci 311 (2007) 400-406. 40. A.A. Khan Behlol, M. Mariappan, A. Veerappan, Spectrochim Acta A Mol Biomol Spectrosc. 137 (2015) 1292-1297. 41. H.I. Jung, S.J. Bowden, A. Cooper, R.N. Perham, Protein Sci 11 (2002) 1091. 42. J. Tian, S. Wei, Y. Zhao, R. Li, S. Zhao, J. Chem. Sci 122 (2010) 391-400. 43. D.P. Ross, S. Subramanian, Biochemistry 20 (1981) 3096–3102. 44. N.A.M. Sultan, B.G. Maiya, M.J. Swamy, Eur. J. Biochem. 271 (2004) 3274–3282. 45. M. Goel, D. Jain, K.J. Kaur, R. Kenoth, B.G. Maiya, M.J. Swamy, D.M. Salunke, J. Biol. Chem 276 (2001) 39277–39281. 46. F.L. Cui, J.L. Wang, Y.R. Cui, J.P. Li, Anal. Chim. Acta 571 (2006) 175 47. J.N. Tian, J.Q. Liu, W.Y. He, Z.D. Hu, X.J. X.G. Chen, Biomacromolecules 5 (2004) 1956-1961. 48. T. Yuan, A.M. Weljie, H.J. Vogel, Biochemistry 37 (1998) 3187-3195. 18

49. P. Padma, S.S. Komath, S.K. Nadimpalli, M.J. Swamy, Phytochemistry 50 (1999) 363– 371. 50. I.J. Goldstein, R.D. Poretz, Academic Press, New York, USA 33 (1986). 51. D.D. Roberts, I.J. Goldstein, J. Biol. Chem 257 (1982) 11274-11277. 52. D.D. Roberts, I.J. Goldstein, Arch. Biochem. Biophys 224 (1983) 479-484. 53. M.J. Maliarik, I.J. Goldstein, J. Biol. Chem 263 (1988) 11274-11279. 54. C.V. Gegg, D.D. Roberts, I.H. Segel, M.E. Etzler, Biochem 31 (1993) 6938-6942. 55. T. Kitao, K. Hattori, Nature 265 (1977) 81 56. D.G. Gilliland, R.J. Collier, J.M. Moehring, T.J. Moehring, Proc. Natl. Acad. Sci. USA 75 (1978) 5319 57. T. Yamaguchi, R. Kato, M. Beppu, T. Terao, Y. Inoue, Y. Ikawa, T. Osawa, J. Natl. Cancer Inst 62 (6) (1979) 1387-1395.

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Table 1: Number of binding sites (n), association constant (Ka), and thermodynamics parameters associated with SGSL-ZnS-QDs interaction. Temp

n

(K)

Ka

–∆G0

–∆H0

– ∆S0

(× 10-4 M-1)

(kJ mol-1)

(kJ mol-1)

(J mol-1 K-1)

51.97

40.15

288

1.06

3.76 ± 0.19

25.22 ± 0.12

293

1.17

2.70 ± 0.10

24.86 ± 0.09

298

1.04

2.07 ± 0.14

24.61± 0.17

303

1.05

1.56 ± 0.31

24.29 ± 0.50

308

0.98

1.27 ± 0.15

24.19 ± 0.05

20

Figure legends Figure 1: Absorption and fluorescence spectra of ZnS-QDs. Fluorescence spectra were measured by exciting the QDs at 360 nm. Inset corresponds to digital photograph of ZnS-QDs at room light (A) and illuminated with UV lamp at 360 nm (B). Figure 2: XRD pattern of ZnS-QDs. Figure 3: Transmission electron micrograph of the pectin capped QDs. (A) TEM image of ZnSQDs, inset corresponds to particle size distribution obtained from TEM image, (B) HRTEM image of ZnS-QDs. Inset in (B) corresponds to the SAED pattern. Figure 4: (A) SGSL fluorescence emission spectra monitored after addition of ZnS-QDs. The upper spectrum in each panel corresponds to free SGSL and the remaining spectra with decreasing fluorescence emission intensity were obtained in the presence of increasing QDs concentrations. Excitation wavelength = 280 nm (B) Stern-Volmer plots of SGSL fluorescence quenched by ZnS-QDs. Stern-Volmer equation, F0/Fc = 1 + Ksv [Q], where Ksv is Stern-Volmer quenching constant, [Q] is the concentration of ZnS-QDs. Figure 5: (A) Binding curve, ∆F vs ZnS-QDs. Binding curve was obtained by the rectangular hyperbolic function using Origin 8 software. (B) Double logarithmic plot for the interaction of ZnS-QDs with SGSL. A plot of log [(F0-Fc/Fc-F∞)] against log [ZnS-QDs] was analyzed according to [11]. Temp. = 30 oC. The abscissa intercept of this plot yielded the pKa value of the SGSL–ZnS-QDs interaction. Inset: plot of 1/∆F as function of the reciprocal ZnS-QDs concentration. The reciprocal of the Y-intercept of this plot gave the value of ∆F∞, the change in fluorescence intensity, when all the protein molecules are bound by the ZnS-QDs. Figure 6: Van’t Hoff plots for the interaction of ZnS-QDs with SGSL.

21

Figure 7: Synchronous fluorescence spectra of SGSL in the influence of various concentrations of ZnS-QDs, (A) ∆λ = 30 nm; (B) ∆λ = 80nm. The concentration of SGSL was fixed at 2.5 µM.

22

6000 A

5000

1.5

4000 3000

1.0

2000 0.5

0.0 200

1000

300

400

500

Fluorescence (a.u.)

Absorbance (a.u.)

2.0

B

0 600

Wavelength (nm)

Figure 1 Khan Behlol et al.,

1

Figure 2 Khan Behlol et al.,

2

(A)

(B)

Distribution of particles

40 35 30 25 20 15 10 5 0 1

2

3

4

5

6

Size (nm)

Figure 3 Khan Behlol et al.,

3

(B) 2.0

F0/Fc

Fluorescence (a.u.)

(A)

1.6

1.2

300

350

400

450

500

0

20

40

60

80

[Zns QDs] (µm)

Wavelength (nm)

Figure 4 Khan Behlol et al.,

4

∆F

2000 1500 1000 500

(B)

0.0

-0.4 0.003

0.002

1/∆ F

log[(F0 - Fc)/(Fc - Fα)]

2500

0.4

(A)

0.001

-0.8 0.000 0.00

0.05

0.10

0.15 -1

1/[ZnS QDs] (µ M )

0 0

20

40

60

80

-5.2

[ZnS QDs] (µM)

-4.8

-4.4

-4.0

log [ZnS QDs] Figure 5 Khan Behlol et al.,

5

10.5

ln(KaT)

10.2

9.9

9.6

9.3 3.25

3.30

3.35

3.40

3.45

3.50

-3

1/T (x 10 ) (K) Figure 6 Khan Behlol et al.,

6

(B)

(A)

Figure 7 Khan Behlol et al.,

7

Synthesis and characterization of zinc sulphide quantum dots and their interaction with snake gourd (Trichosanthes anguina) seed lectin Khan Behlol Ayaz Ahmed, Pichaikkannu Ahalya, S. Megarajan, Ravikanth Kamlekar and Veerappan Anbazhagan* Department of Chemistry, School of Chemical and Biotechnology, SASTRA University, Thanjavur, Tamil Nadu, India.

Graphical Abstract

+

ZnS QDs ZnS QDs

SGSL

8

Highlights •

A facile method to synthesize pectin stabilized zinc sulphide quantum dots.

•

Zinc sulphide QDs interacts with snake gourd seed lectin.

•

Interactions are governed by enthalpic forces with negative entropic contribution.

•

Microenvironments around the tryptophan residues of SGSL are perturbed by ZnS QDs.

9