Journal of Luminescence 141 (2013) 53–59 Contents lists available at SciVerse ScienceDirect Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin Conjugation of nano and quantum materials with bovine serum albumin (BSA) to study their biological potential Suman Singh a,n, Rajnish Kaur a, Jitender Chahal a, P. Devi a, D.V.S. Jain b, M.L. Singla a a b Central Scientific Instruments Organisation (CSIR-CSIO), Chandigarh, India Panjab University, Chandigarh, India art ic l e i nf o a b s t r a c t Article history: Received 11 July 2012 Received in revised form 5 February 2013 Accepted 28 February 2013 Available online 14 March 2013 Conjugates of gold nanoparticles (AuNPs) and semiconductor quantum dots (CdS/T) have been synthesized with bovine serum albumin (BSA) using wet chemistry. The optical properties of nano and quantum materials and their BSA conjugate have been studied using UV–Visible and Fluorescence spectroscopy. UV–Visible spectrum of pure BSA showed an absorption maximum at 278 nm, which showed blue shift after its conjugation with nano and quantum materials. Increased concentration of AuNPs during conjugation resulted in broadening of BSA peak (278 nm), which can be related to the formation of ground state complex formation, caused by the partial adsorption of BSA on the surface of NPs. However, increased concentrations of BSA resulted in decrease in SPR intensity of gold nanoparticles (528 nm) and absorbance peak of BSA started diminishing. AuNPs acted as quencher for BSA fluorescence intensity, when excited at 280 nm. The binding constant (K) and the number of binding sites (n) between AuNPs and BSA have been found to be 1.97  102 LM−1 and 0.6 respectively. With quantum dots, conjugation resulted in enhancement of fluorescence emission of quantum dots when excited at 300 nm, which might be due to the stabilizing effect of BSA on QDs or due to energy transfer from tryptophan moieties of albumin to quantum dots. & 2013 Elsevier B.V. All rights reserved. Keywords: BSA CdS quantum dots Gold nanoparticles 1. Introduction Nanoparticles (NPs) have significant adsorption capacities due to their relatively large surface area, therefore they are able to bind or carry other molecules such as chemical compounds, drugs, probes and proteins attached to their surface by covalent bonds or by adsorption. Hence, the physicochemical properties of nanoparticles, such as charge and hydrophobicity, can be altered by attaching specific chemical compounds, peptides or proteins to the surface [1,2]. Adsorption of protein molecules on NP surface changes their surface functionality, thus influencing their behavior in biological systems. Moreover, formation of NP–protein conjugates provides stability to NPs over broad range of pH and ionic strengths. Smaller nanoparticles favor native-like protein structure more strongly, resulting in higher intrinsic enzyme activity [3], whereas larger nanoparticles provide larger surface area of contact for adsorbed proteins and thus results in stronger interactions between proteins and nanoparticles. The efficiency of this interaction can be a decisive factor for the fate of a nanoparticle within a biological system. But at the same time, the interaction between n Corresponding author. Tel.: +91 172 265 17 87; fax: +91 172 265 70 82. E-mail addresses: sumansingh01@gmail.com (S. Singh), singla_min@yahoo.co.in (M.L. Singla). 0022-2313/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jlumin.2013.02.042 NPs and protein leads to greater perturbation of protein structure. Many studies have found that loss of α-helical content occurs when proteins are adsorbed onto nanoparticles, with or without an increase in β-sheet [4,5]. However, Ganguly et al. [6] in their studies found that though some conformational changes take place in bio-nanoconjugate in the presence of cadmium sulfide NPs, α-helix structure retained its identity. Owing to high extinction coefficient and a broad absorption spectrum in visible region, gold nanoparticles (NPs) have been of great interest [7]. Due to their size dependent properties and dimensional similarities with biological macromolecules, these nanomaterials show potential application for biochemical studies. Thus, the study of biological effects of NPs have gained momentum and many studies have been reported on the interaction of nanoparticles and their conjugates with species of biological origin [8–10]. Adsorption and conformation of serum albumin protein on gold nanoparticles have been investigated by Hackle et. al. [11]. Effect of surface functionalities of NPs on their interaction with BSA has been studied by Treuel et. al. [12]. Luminescent quantum dots, another class of nanomaterials, are also equally valued for their unique optical properties. The wide excitation and narrow emission wavelengths, resistance to photobleaching, and high quantum yield make them attractive for biological applications, thus resulting in extensive concern about its biological toxicity [13,14]. Klostranec and Chan reviewed the 54 S. Singh et al. / Journal of Luminescence 141 (2013) 53–59 role of quantum dots in biological and biomedical applications and critically evaluated the barriers impacting current quantum dot technologies [15]. Bovine serum albumin (BSA) is a commonly studied protein for investigating the effect of nanoparticles on protein conformational change. Pure BSA exhibits an absorption maximum at 278 nm, which originates from the aromatic residues tryptophan and tyrosine and the disulfide bonds in the protein. Since BSA is rich in amino moieties and also contains several disulfide bonds with one free thiol in the cysteine residues [16], these functionalities help them to conjugate with nanomaterials via thiolate linkage and/or weak covalent bonds with alkylamines, or through electrostatic interactions. Herein, we studied the interaction of bovine serum albumin (BSA) protein with gold nanoparticles and CdS quantum dots using UV–Visible spectroscopy and fluorescence spectroscopy. 2. Experimental 2.1. Materials Cadmium nitrate tetrahydrate [Cd(NO3)2d4H2O], and sodium sulfide [Na2SdXH2O] flakes were procured from Merck private limited, dimethyl sulphoxide (DMSO) of GC grade and tetra chloro auric acid were procured from Spectrochem. Pvt. Ltd., Mumbai. Mercaptoacetic acid (MPA) was from Sisco Research Laboratories Pvt. Ltd., and bovine serum albumin (BSA), purchased from Sigma, was used without further purification. The stock solution of BSA (10 mg/ml) was prepared in 1 mM sodium acetate buffer of pH 3.5. Sodium sulfide, tetra auric acid and hydrazine hydrate solutions were prepared in DI water. A series of bioconjugates with different molar ratios of BSA/AuNPs and BSA/QDs were prepared by keeping the concentration of BSA constant while varying the concentration of NPs and QDs and vice-versa. All the measurements in the following discussion were performed at room temperature. 2.2. Instrumentation UV–Visible spectra of AuNPs, AuNPs-BSA conjugate, QDs and QDs-BSA conjugate were recorded on Hitachi U-3400 UV–Visible spectrophotometer. Fluorescence spectra of these nanomaterials were recorded on Cary Eclipse fluorescence spectrophotometer (Varian), using excitation and emission slit of 5 nm. A 1.00 cm path length rectangular quartz cell was used for UV–Vis and fluorescence studies. Raman spectra were recorded on Renishaw (UK) Raman Analyzer. The transmission electron microscopy (TEM) images of the QDs and AuNPs are acquired on a JEOL-JEM-2010 transmission electron microscope (Japan). Zeta Potential measurements of QDs and their BSA conjugate were carried out on Malvern (USA) Zeta Sizer. Very dilute solutions of AuNPs, QDs and BSA were used in the experiment to avoid inner filter effects and selfadsorption. 2.3. Synthesis of gold nanoparticles (AuNPs) AuNPs were synthesized using ascorbic acid as reducing agent. For synthesis 1% solution of auric acid was brought to boiling and 1 mM of ascorbic acid was added slowly. The color of solution slowly turned from pale yellow to pinkish red. The role of ascorbic acid concentration plays crucial role in controlling the size of NPs. Higher concentrations lead to the formation of multi-shaped NPs. Thus in present studies, low concentrations have been used. The solution was cooled at room temperature and measured its absorbance using UV–Visible spectrophotometer. 2.4. Synthesis of CdS quantum dots CdS QDs have been synthesized using wet chemistry which involved dissolving cadmium nitrate tetrahydrate (0.05 M), used as precursor, in degassed dimethyl sulphoxide followed with vigorous stirring (1000 rpm) for about 20 min at a reaction temperature of 70 1C. This was followed by the addition of a surfactant (thioglycolic acid) to cap quantum dots (CdS/T). After vigorous stirring of 30 min, 10 ml of sodium sulfide was added slowly as a precursor of sulfide ions which resulted in the formation of precipitate. The precipitate was filtered and washed properly with distilled water and ethanol through centrifugation at 8000 rpm for 20 min. To avoid the problems of toxicity and photochemical instability, CdS QDs were further functionalized with polyethylene glycol (PEG) (CdS/T-PEG). Scheme 1 shows pictorial representation of QDs synthesis. 2.5. Conjugation of AuNPs and CdS/T-PEG QDS with protein In our studies, BSA has been used as role protein. BSA solution (1 mM) was prepared in sodium acetate buffer (pH 3.5). Mixture was allowed to incubate for 1 h at room temperature with continuous mild shaking. Incubation was followed with centrifugation at 1500 rpm for about 20 min, which allows the removal of any unbound nanoparticle or protein molecule. Scheme 2 shows pictorial representation of AuNPs-BSA conjugate synthesis. Similar procedure was used for the preparation of QDs-BSA conjugate. 3. Results and discussion 3.1. TEM studies Fig. 1A shows TEM image of gold nanoparticles which are spherical in shape with size of approximately 12 nm. Fig. 1B shows TEM image of spherical CdS QDs, having size of approximately between 4 and 7 nm. Arrows referred towards the QDs of size 4 nm. Scheme 1. Synthetic route of CdS/T-PEG QDs. S. Singh et al. / Journal of Luminescence 141 (2013) 53–59 55 Scheme 2. Pictorial representation of AuNPs-BSA conjugate synthesis. by an interacting electromagnetic field. The average size of nanoparticles comes out to be 16 73 nm. To study the effect of conjugation on absorption properties of BSA and gold nanoparticles, absorption spectra of native BSA and AuNPs-BSA conjugate were recorded (Fig. 2b). Spectra were also recorded with varied concentrations of BSA and gold nanoparticles. Pure BSA exhibits an absorption maximum at 278 nm, which originates from the aromatic residues tryptophan and tyrosine and the disulfide bonds in the protein. After conjugation with gold nanoparticles, the absorption bond of BSA showed blue shift of about 20 nm, with a strong plasmon absorption peak at 530 nm attributed to AuNPs collective electron oscillations or localized surface plasma resonance (SPR). Increased concentration of AuNPs for conjugation resulted in increased intensity of absorption peak of tryptophan at 278 which however started to change into a broad hump (Fig. 2c). The appearance of hump can be related to the interaction between serum albumin and AuNPs through a ground state complex formation, caused by the partial adsorption of BSA on the surface of NPs [17,18]. Such an adsorption leads to a partial unfolding of BSA due to the breaking of disulfide bridges in view of the covalent interactions of cysteine residues with NP surface thus causing conformational changes [19–21]. Increasing the concentration of BSA during conjugation however resulted in decreased intensity of SPR band of AuNPs (Fig. 2d). Fig. 1. TEM image of (A) AuNPs (B) CdS QDs. 3.2.2. CdS quantum dots and conjugate with BSA Absorbance studies were also carried out for CdS/T-PEG quantum dots and it BSA conjugate (Fig. 2e). For BSA, absorbance peak was observed at 278 nm, which in case of its conjugate with quantum dots showed blue shift of about 25 nm. Also, the absorbance band corresponding to CdS/T-PEG QDs started disappearing with increase in BSA concentration, confirming the strong bonding between quantum dots and BSA through covalent interactions of cysteine residues with QDs surface. 3.3. Stability of AuNPs 3.2. Absorbance Studies 3.2.1. AuNPs and its protein conjugate The absorbance was measured for gold solution, ascorbic acid and colloidal gold (AuNPs) (Fig. 2a). Auric acid and ascorbic acid did not show any absorbance in the covered range. The synthesized gold nanoparticles showed absorbance at 530 nm arising from the collective oscillation of free conduction electrons induced The stability of resultant AuNPs have been checked using conventional electrolyte introducing method, where we added different concentrations of NaCl (0.25 M to 4.0 M) to AuNPs solution and measured the absorption maxima (data not shown). No shift in λmax was observed from 0.25 M to 2.0 M of NaCl. This shows that gold colloid solution is quite stable in the presence of these concentrations of NaCl. But from 2.5 M to 4.0 M, SPR band position showed red shift (from 527 to 533 nm), along with its broadening, which might be due 56 S. Singh et al. / Journal of Luminescence 141 (2013) 53–59 Fig. 2. (a): UV–Visible spectrum of AuNPs, (b): Absorbance spectra of pure BSA and AuNPs-BSA conjugate, (c): Absorbance spectra of AuNPs-BSA conjugate with increasing concentration of AuNPs (0.0 to 0.033  10−8 M), (d): Absorbance spectra of pure BSA (a) pure AuNPs (b) and AuNPs-BSA conjugate with varied BSA concentration (c) and (e): Absorbance spectra of CdS/T-PEG QDs and CdS/T-PEG/BSA conjugate. to the formation of unstable colloids at high ionic strength of NaCl. The red shift can be explained in terms of electric double layer thickness which stabilizes nanoparticle by repulsing other particles. This electrostatic stabilization is very susceptible to the presence of salts in the medium, whose thickness decreases as the concentration of electrolyte increases [22]. Since ion strength determines the debye length around the particles, it will change the mean closest approach distance between gold nanoparticles, resulting in aggregation of nanoparticles [23,24]. The AuNPs-BSA conjugate however showed no change in λmax on adding these concentrations of NaCl electrolyte, which shows that conjugate is quite stable against the electrolyte. Scheme 3 shows pictorial representation of role of NaCl on the stability of AuNPs. 3.4. Raman spectra of BSA and AuNPs-BSA conjugate Fig. 3 shows the Raman spectra recorded for AuNPs and its conjugate with BSA. The presence of stretching vibration of disulfide bridges at 519 cm−1 in the SERS spectrum of AuNPs-BSA is a strong indicator that the disulfide bonds remain unbroken and the protein do not denature at room temperature when adsorbed to AuNPs. The C–H3 & C–H2 deformation vibrations from the side chains of different A. A arise in the range of 1440–1480 cm−1. Band at 1684 cm−1 arise from CQO, and C–N stretching. Bands in the range 1650–1700 cm−1 are assigned to β-turn structure. Much difference is not observed between spectrum of BSA and AuNPsBSA bioconjugate due to similarities in their functional groups. 3.5. Fluorescence studies 3.5.1. AuNPs-BSA conjugate Since the fluorescence of tryptophan residues of BSA is sensitive to their vicinity, we have monitored the fluorescence quenching of tryptophan residue in presence of quencher (AuNPs) (Fig. 4a). Increase in the concentration of AuNPs led to linear quenching S. Singh et al. / Journal of Luminescence 141 (2013) 53–59 57 Scheme 3. Pictorial representation of effect of NaCl on AuNPs and its conjugate with BSA. concentration using following equation: FO ¼ 1 þ K q τ0 ½Q Š ¼ 1 þ K SV ½Q Š F Fig. 3. Raman spectra of BSA and AuNPs-BSA conjugate. of fluorescence intensity of tryptophan residue of BSA, when excited at 280 nm. 1:1 M ratio of gold nanoparticles and BSA resulted in about 56% quenching of fluorescence intensity of BSA. The nanoparticle shape is however critical in the interaction of gold nanoparticles with biological molecules like BSA. Gold nanorods show enhancement in the fluorescence intensity [25]. This quenching in fluorescence in presence of gold nanoparticles revealed that nanoparticles reside near tryptophan of the BSA protein molecule, which is consistent with an earlier report [4]. Quenching also implies efficient energy transfer between BSA moiety and gold nanoparticles [4]. Quenching in presence of gold nanoparticle takes place mainly through a static quenching mechanism, which arises from the formation of complex between BSA and Au nanoparticles [17]. This complex formation is facilitated due to the presence of amino moieties in BSA that contains disulfide bonds with free thiol [16]. Stern–Volmer relation [26] has been used to explain the relationship between quenching of excited states and gold nanoparticle ð1:0Þ Fo and F are relative fluorescence intensity in absence and presence of gold nanoparticles (quencher), Kq is bimolecular quenching rate constant, τ0 is average lifetime of fluorophore in absence of quencher, and [Q] is concentration of quencher, KSV is Stern–Volmer quenching constant. Fig. 4b shows the Stern–Volmer plot of Fo/F versus gold nanoparticle concentration [AuNPs]. The plot showed positive deviation (upward) from linearity after about 2.0  10−10M concentration of AuNPs which suggests that both static and dynamic quenching are simultaneously involved [27]. At lower concentrations, static quenching was dominant but as the concentration of quencher increased, both dynamic and static quenching acted. To further understand the quenching, graph was plotted between [1−(F/Fo)]/[Q] and F/Fo (Fig. 4c), where [Q] is the concentration of quencher (AuNPs), F is the fluorescence in presence of quencher, Fo is the fluorescence in absence of quencher. From the slope and intercept of the plot, dynamic and static quenching constants were calculated and value was 1.3  106 and 1.5  107 for dynamic quenching and static quenching respectively. These values suggest the dominance of static quenching. This might be due to the fact that initially quencher molecule happens to be randomly positioned in the proximity of fluorescent molecule at the time its excitement. So only a fraction of molecules are actually quenched during the collision process. The remaining molecules in excited state are deactivated instantaneously after being excited since quencher molecule is situated in the proximity of the excited molecules and interacts with them. The probability of the quencher to be found in the sphere of action with a volume V depends on the volume V and on the quencher concentration [28]. However, when the concentration of quencher is more, molecules available for collision also increases hence dynamic quenching also comes into an action at higher concentrations. Further, the absorption spectrum (Fig. 2b) of BSA showed blue shift of about 20 nm in the presence of AuNPs which might be due to the greater percentage of static quenching [29]. Further, static and dynamic quenching can be differentiated by their dependence on temperature and viscosity. Fig. 4d shows 58 S. Singh et al. / Journal of Luminescence 141 (2013) 53–59 Fig. 4. (a) Fluorescence spectra of BSA in the presence of gold nanoparticles (0.0 to 0.044  10−8 M), (b): Stern-Volmer plot of AuNPs-BSA conjugate (AuNPs concentration from 0.0 to 0.044  10−8 M), (c): Plot of [1−(F/Fo)]/[Q] Vs F/Fo, (d): Stern-Volmer plot of AuNPs-BSA conjugate as a function of temperature and (e): plot of log[(Fo-F/F-Fsat)] versus log[AuNPs concentration, M] Stern–Volmer plot as a function of temperature. At 25 1C, 35 1C, 45 1C and 55 1C, the plot continued with positive deviation but at higher temperatures (65 1C & 75 1C), the plot showed linear relationship. This behavior also shows the presence of both static and dynamic quenching, but since at higher temperature, collision rate increases, the chances of collision between flurophore and quencher becomes high. 3.5.1.1. Binding constants and number of binding sites. For static quenching process, Tedesco model has been used for the determination of binding constant (K) and the number of binding sites (n) using the relationship [30,31]: F o −F ¼ ð½Q Š=KdissÞn F−Fsat Here Fo denotes fluorescence intensity of fluorophore in absence of quencher, Fsat represents the fluorescence intensity of fluorophore at the highest concentration of quencher, F refers to the fluorescence intensity of intermediate concentrations between Fo and Fsat. [Q] is the concentration of quencher, n is the number of binding sites per particles and Kdiss is the reciprocal of binding constant (K). The values of ‘n’ and ‘K’ were determined from the slope and intercept of plot of log[(Fo−F/F−Fsat)] versus log[AuNPs concentration] shown in Fig. 4e and values are 0. 75 for ‘n’ and 0.13 LM−1 for binding constant (K). 3.5.2. Quantum dots and BSA conjugate 3.5.2.1. Fluorescence of CdS QDs. Fluorescence behavior of CdS/T and CdS/T-PEG quantum dots has been studied (Fig. 5a). The QDs showed emission at 515 nm which is somewhat blue-shifted compared to bulk CdS at 650 nm [32]. Quantum dots coated with PEG showed about 20% enhancement in fluorescence intensity. PEG, forms the hydrophilic shell layer of tethered chains on the gold surface, increasing the dispersion of the particle stability in aqueous media via a steric repulsion effect. 3.5.2.2. Fluorescence study of CdS/T-PEG/BSA conjugate. When pegylated quantum dots (CdS/T-PEG) were conjugated with BSA, reverse behavior was observed as compared to AuNPs-BSA conjugate. The emission intensity of quantum dots showed strong enhancement on the addition of BSA from 5 mM to 90 mM, thereafter it became constant, along with shift in emission wavelength towards shorter value (blue shift) (Fig. 5b). Further additions of BSA (higher concentrations) however resulted in quenching of fluorescence emission (data not shown). The increase of fluorescence intensity of quantum dots can be explained in terms of passivation of defects on their surface or the energy transfer from tryptophan moieties of albumin to quantum dots, which is analogous to classical förster energy transfer. The result is similar to the previous reports [9,13]. At the same time, this increase in emission might be also due to the stabilizing effect of BSA on quantum dots. To confirm this, we carried out zeta potential measurements of QDs and QDs-BSA conjugate (Fig. 5c) and it was observed that after conjugation, zeta potential of CdS/TPEG QDs increased from −0.042 mV to −30.5 mV, which supports the stabilizing effect of BSA on QDs. 4. Conclusion The interaction of nano and quantum materials with biomolecules recently attracted increasing attention owing to their wide application in biological areas. In this study, optical methods (UV– vis and fluorescence spectroscopy) have been used to investigate the interaction of gold nanoparticles and thiol capped CdS quantum dots with BSA protein. With AuNPs, interaction resulted in quenching effect on the emission intensity of BSA. The positive deviation in Stern–Volmer plot reflected the presence of both static and dynamic quenching. The values of binding constant and number of binding sites have been found to be 1.97  102 LM−1 and 0.6 respectively. For conjugate with quantum dots, BSA resulted in enhancement of emission intensity of QDs which shows that here protein acted as stabilizer. S. Singh et al. / Journal of Luminescence 141 (2013) 53–59 59 Fig. 5. (a): Emission spectra of CdS/T and CdS/T-PEG quantum dots, (b): Emission Spectra of CdS/T-PEG as a function of increased BSA concentration (bottom to top) and (c): Zeta potential determination of CdS/T-PEG QDs and its conjugate with BSA. Acknowledgment Authors acknowledge the financial support from the Council of Scientific and Industrial Research (CSIR), New Delhi for financial support under XIIth five year plan. Authors are also thankful to Director, CSIR-CSIO, Chandigarh for infrastructure facility. References [1] L. Crombez, M.C. Morris, S. Deshayes, F. Heitz, G. Divita, Curr. Pharm. Des. 14 (2008) 3656. [2] D. Aili, K. Enander, J. Rydberg, I. Nesterenko, F. Björefors, L. Baltzer, B. Liedberg, J. Am. Chem. Soc. 130 (2008) 5780. [3] A.A. Vertegel, R.W. Siegel, J.S. Dordick, Langmuir 20 (2004) 6800. [4] L. Shang, Y. Wang, J. Jiang, S. Dong, Langmuir 23 (2007) 2714. [5] X. Wu, G. Narsimhan, Biochim. Biophys. Acta (BBA) 1784 (2008) 1694. [6] G. Mandal, M. Bardhan, T. Ganguly, J. Phys. Chem. C 115 (2011) 20840. [7] J.-S. Choi, Y.-W. Jun, S.-I. Yeon, H.C. Kim, J.-S. Shin, J. Cheon, J. Am. Chem. Soc. 128 (2006) 15982. [8] L. Yang, R. Xing, Q. Shen, K. Jiang, F. Ye, J. Wang, Q. Ren, J. Phys. Chem. B 110 (2006) 10534. [9] N.N. Mamedova, N.A. Kotov, A.L. Rogach, J. Studer, Nano Lett. 1 (2001) 281. [10] J.L. Burt, C. Gutiérrez-Wing, M. Miki-Yoshida, M. José-Yacamán, Langmuir 20 (2004) 11778. [11] D.-H. Tsai, F.W. DelRio, A.M. Keene, K.M. Tyner, R.I. MacCuspie, T.J. Cho, M. R. Zachariah, V.A. Hackley, Langmuir 27 (2011) 2464. [12] L. Treuel, M. Malissek, J.S. Gebauer, R. Zellner, ChemPhysChem 11 (2010) 3093. [13] S.X.D. Yang, Q. Chen, Y. Wang, Colloids Surf. A 329 (2008) 38. [14] R.B.T. Jamieson, D. Petrova, R. Pocock, M. Imani, A.M. Seifalian, Biomaterials 28 (2007) 4717. [15] J.M. Klostranec, W.C.W. Chan, Adv. Mater. 18 (2006) 1953. [16] M.J. Meziani, P. Pathak, B.A. Harruff, R. Hurezeanu, Y.-P. Sun, Langmuir 21 (2005) 2008. [17] P.B. Kandagal, S. Ashoka, J. Seetharamappa, S.M.T. Shaikh, Y. Jadegoud, O.B. Ijare, J. Pharm. Biomed. Anal. 41 (2006) 393. [18] Y.-Q. Wang, H.-M. Zhang, G.-C. Zhang, W.-H. Tao, Z.-H. Fei, Z.-T. Liu, J. Pharm. Biomed. Anal. 43 (2007) 1869. [19] D. Zhang, O. Neumann, H. Wang, V.M. Yuwono, A. Barhoumi, M. Perham, J.D. Hartgerink, P. Wittung-Stafshede, N.J. Halas, Nano Lett. 9 (2009) 666. [20] C.D. Keating, K.M. Kovaleski, M.J. Natan, J. Phys. Chem. B 102 (1998) 9404. [21] S. Chah, M.R. Hammond, R.N. Zare, Chem. Biol. 12 (2005) 323. [22] H.W.D. Fennell Evans, The Colloidal Domain: Where Physics, Chemistry, Biology, and Technology Meet (Advances in Interfacial Engineering), WileyVCH, New York, 1999. [23] J.N. Israelachvili, Intermolecular and Surface Forces, 2nd, Academic Press, 1991. [24] R.J. Hunter, Foundation of Colloid Science, Claredon Press, Oxford, USA, 1993. [25] B. Pan, D. Cui, P. Xu, Q. Li, T. Huang, R. He, F. Gao, Colloids Surf. A 295 (2007) 217. [26] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed, Kluwer Academic/Plenum Publishers, New York, 1999. [27] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed., Springer, New York, 2006. [28] R.I.T.A. Airinei, E. Rusu, D.O. Dorohoi, Digest J. Nanomater. Biostruct. 6 (2011) 1265. [29] S. Bhattacharyya, T. Sen, A. Patra, J. Phys. Chem. C 114 (2010) 11787. [30] P. Kumar, A. Deep, S.C. Sharma, L.M. Bharadwaj, Anal. Biochem. 421 (2012) 285. [31] A.C. Tedesco, D.M. Oliveira, Z.G.M. Lacava, R.B. Azevedo, E.C.D. Lima, P. C. Morais, J. Magn. Magn. Mater. 272–276 (Part 3) (2004) 2404. [32] Y.C. Cao, J. Wang, J. Am. Chem. Soc. 126 (2004) 14336.