Insights into the binding of photothermal therapeutic agent bismuth sulfide nanorods with human serum albumin

Abstract

The biomedical application of bismuth sulfide (Bi₂S₃) nanorods as a nanomedicine for the photothermal therapy of tumors prompted this study into their interactions with human serum albumin (HSA) to understand their pharmacokinetics. Bi₂S₃ nanorods with orthorhombic crystalline structure were synthesized using a simple microwave irradiation method from bismuth nitrate, sodium sulfide, and starch in an aqueous medium. The synthesized Bi₂S₃ nanorods and their formation mechanism were well-characterized by powder X-ray diffraction (XRD), high-resolution transmission electron microscopy (HRTEM), selected area electron diffraction (SAED), and energy-dispersive X-ray (EDX). The interactions of the Bi₂S₃ nanorods with HSA was investigated by absorption spectroscopy, fluorescence spectroscopy and circular dichroism spectroscopy. The absorption and time-resolved fluorescence spectroscopy studies confirmed that the Bi₂S₃ nanorods interacted with HSA through a static mechanism. The steady-state and synchronous fluorescence spectral studies showed that the single binding site is near the tryptophan moiety (Trp-214) in HSA. The moderate binding constant determined from the steady-state fluorescence study suggested the possibility of effective transportation of Bi₂S₃ nanorods inside the body. These results could be very helpful for understanding the mechanisms and pathways responsible for the uptake, distribution and catabolism of Bi₂S₃ nanorods in multiple tissues of the human body.

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1. Introduction

Semiconductor nanomaterials are attracting an overwhelming attention for their potential applications especially in the fabrication of optical and electronic devices.¹ Of these materials, bismuth sulfide (Bi₂S₃), which is a semiconductor with low toxicity and low direct band gap,^2–4 has attracted considerable interest due to its potential applications in thermoelectric devices, electronic devices, optoelectronic devices, electrochemical hydrogen storage, hydrogen sensors, biosensors, photocatalytic hydrogen production, photocatalytic degradation, supercapacitors and biomolecular detection.^6–10 One-dimensional (1-D) nanostructured Bi₂S₃ materials, such as nanorods, nanotubes, nanoribbons, nanocables and nanowires, have attracted considerable interests because of their unusual properties facilitating efficient electron transfer and potential uses in the development of nanodevices.^11–13 To synthesize 1-D Bi₂S₃ nanostructures, many techniques, including single source precursor methods, evaporation route, thermal decomposition, hydrothermal method, solvothermal route, solventless thermolysis method, biomolecule-assisted approach, microwave irradiation, sonochemical method and template route have been utilized.^12,14,15 Among them, microwave irradiation has more advantages such as shorter reaction time, higher reaction rate, more selective products with high yield, easy controllability, and less cost. In addition, due to the uniform heating, this method facilitates the generation of smaller particles with a narrow size distribution and high phase purity.^1,16

Liu et al.⁴ found that Bi₂S₃ nanorods can serve as nanomedicine for in vivo multimodal imaging-guided photothermal therapy of tumor. However, to utilize it as a nanomedical device, understanding their pharmacokinetics is needed but these results are still awaited. To understand their pharmacokinetics, the interaction of Bi₂S₃ nanorods with human serum albumin (HSA) needs to be investigated because HSA transports the bioactive substances in the circulatory system as a carrier conjugate and the pharmacokinetics depends on the binding affinity of the bioactive substances with HSA.^17,18 This in vitro binding interaction between bioactive substances and HSA can be identified easily using spectral methods. In particular, the fluorescence quenching technique is the most frequently used method to monitor the molecular interactions because of its high sensitivity, reproducibility, and relatively very easy.¹⁹

HSA is the most abundant extracellular protein in the circulatory system of the human body, which not only transports endogenous and exogenous compounds and a variety of pharmaceuticals, but also regulates the colloidal osmotic pressure of blood.^20,21 While binding with a bioactive substances, it increases the solubility and reduces the toxicity of the bioactive substances and protects the bound bioactive substance from oxidation.¹⁸ It is synthesized by the human liver and its half-life is approximately 19 days. It is a heart shaped molecule consisting of a single non-glycosylated polypeptide chain with 585 amino acid residues restrained by disulfide bridges, and a molecular weight of about 66.438 kDa.^21,22 It comprised of three homologous alpha-helical domains I (1–195), II (196–383) and III (384–585) that are packed in two separate sub-domains A and B, which contain six and four α-helices, respectively.^21,23 According to Sudlow's nomenclature, it has two principal binding sites, namely, Sudlow's I and II sites, which are located in subdomain IIA and IIIA, respectively.²¹

This study examined the interaction of Bi₂S₃ nanorods with HSA (Fig. 1a) to assess their binding characteristics to understand their pharmacokinetics. In this regard, Bi₂S₃ nanorods were synthesized successfully by simple microwave irradiation method using bismuth nitrate and sodium sulfide as bismuth and sulfur source, respectively, along with starch as a stabilizing agent. The interaction of the microwave synthesized Bi₂S₃ nanorods with HSA was investigated using different spectroscopic methods such as UV-Vis spectroscopy, fluorescence and circular dichroism (CD). On the basis of the spectroscopy data, the quenching mechanism, binding constant, the number of binding sites, the binding site and the energy transfer process distance of HSA–Bi₂S₃ nanorods were calculated. Furthermore, the secondary and tertiary structural changes that occurred in the HSA on binding with Bi₂S₃ nanorods were estimated using CD spectral analysis. Thus, the obtained results may have significant implications in nanomedical device design.

Fig. 1 (a) Interaction between HSA and Bi₂S₃ nanorods. XRD (b), TEM (c), HRTEM (d), SAED (e), and EDX (f) of Bi₂S₃ nanorods.

2. Materials and methods

2.1 Materials

Bismuth nitrate pentahydrate Bi(NO₃)₃·5H₂O, sodium sulfide nonahydrate Na₂S·9H₂O, and HSA were purchased from Sigma-Aldrich. All other reagents used were of analytical reagent grade and used without further purification. Water from a Milli Q system apparatus (Millipore, USA) was used throughout the experiments.

2.2 Methods

2.2.1 Microwave assisted synthesis of Bi₂S₃ nanorods. Bi(NO₃)₃·5H₂O (10 mM) and starch (1 wt%) were dissolved in 25 ml of water taken in a round-bottomed flask. Under stirring, an aqueous Na₂S·9H₂O (15 mM, 25 ml) solution was added drop-wise to the round-bottomed flask and this mixture was heated for 10 min in a domestic cooking microwave oven (2450 MHz, Whirlpool) with a refluxing apparatus circulated with ice-cold water. The resulting black precipitate was centrifuged, washed several times with distilled water and ethanol, then dried in vacuum at 60 °C and collected for further characterization.

2.2.2 Interaction studies. HSA solution was prepared in pH 7.4 phosphate buffer solution and stored at 0–4 °C. HSA concentration was maintained at 4 μM and the concentration of Bi₂S₃ nanorods was varied from 0 to 4 μM. This mixture was sonicated in an ultrasonic bath for 1 minute to make the serum albumins and Bi₂S₃ nanorods to combine completely. This titration was monitored by steady-state fluorescence, time-resolved fluorescence and UV-Vis absorption spectroscopy. For synchronous fluorescence spectroscopy and circular dichroism technique, the HSA concentration was maintained at 4 μM in the absence (0 μM) and presence (4 μM) of Bi₂S₃ nanorods.

2.3 Instrumentation

The Bi₂S₃ nanorods were characterized by powder X-ray diffraction (XRD), high-resolution transmission electron microscopy (HRTEM), selected area electron diffraction (SAED), and energy-dispersive X-ray (EDX). The XRD patterns were obtained with a Philips PW1710 diffractometer using Ni filtered Cu_α radiation. The sample was allowed to equilibrate with atmospheric moisture for at least 24 hours prior to recording. The scanning range was 10–80° (2θ) with a step of 0.02° and a count time of 2 seconds. TEM images along with SAED pattern and EDX spectrum were obtained in TECNAI G² model, in which the samples were coated on a copper grid at normal atmospheric temperature and pressure.

The interaction was investigated by UV-Vis absorption spectroscopy, fluorescence spectroscopy, synchronous fluorescence spectroscopy and circular dichroism spectroscopy. The UV-Vis absorption spectra were obtained on a T90+ UV-Vis spectrophotometer (PG Instruments, United Kingdom) with 1.0 cm quartz cell. The fluorescence spectroscopy measurements were performed on RF-5301 PC spectrofluorophotometer (SCHIMADZU, Japan) using a 1.0 cm quartz cell. Excitation and emission slit widths of 5 nm were constantly retained for fluorescence studies. The scan speed was maintained at 240 nm min⁻¹. The samples were cautiously degassed using pure nitrogen gas for 15 minutes before every experiment. Fluorescence lifetime measurements were carried out in a picosecond time correlated single photon counting (TCSPC) spectrometer with a tunable Ti-sapphire laser (TSUNAMI, Spectra physics, USA) as the excitation source. The fluorescence decay curves were analyzed using the software provided by IBH (DAS-6). The circular dichroism (CD) spectra were obtained on a Jasco J-810 spectropolarimeter under constant nitrogen flush over a wavelength range of 190–270 nm at a scanning speed of 200 nm min⁻¹.

3. Results and discussion

3.1 Characterization of Bi₂S₃ nanorods

The phase crystallinity and purity of microwave synthesized Bi₂S₃ nanorods were confirmed by the powder XRD (Fig. 1b). All diffraction peaks were readily indexed to the orthorhombic Bi₂S₃ phase of the JCPDS database no. (17-0320). No other peaks were present, which indicates the purity of the Bi₂S₃ nanorods. The sharp diffraction peaks indicates that the products (Bi₂S₃) are well crystalline orthorhombic structure.¹

The morphology and composition of microwave synthesized Bi₂S₃ nanorods was characterized by transmission electron microscopy. The TEM image (Fig. 1c) shows well-crystallized rod-like shaped particles with average dimensions of ca. 15 nm in width and range of 60–100 nm in length. In addition, it showed the random distribution of Bi₂S₃ nanorods, which can be attributed to the very fast growth of Bi₂S₃ in water medium.⁸ Slightly agglomerated particles were evident from the TEM image, which may be due to the drying process of the sample preparation for TEM. High resolution TEM (Fig. 1d) shows the lattice structure with the interplanar spaces of 0.55 nm in parallel with the nanorods, corresponding to the [2 0 0] crystallographic plane. The plane [2 0 0] is parallel to the growth direction, indicating that the nanorod tends to grow along the [0 0 1] direction.⁵ The SAED pattern of the Bi₂S₃ nanorods, shown in Fig. 1e, revealed several diffraction rings, which is the sum of the diffraction pattern of different individual nanorods, indicating the polycrystalline nature.^1,16 The energy dispersive X-ray (EDX) spectrum shown in Fig. 1f suggests that Bi and S were detected in the ratio of 38.5 : 61.5, which is nearly 2 : 3. The other peaks, such as C and O, are ascribed to the presence of starch, which is used as the stabilizer, whereas Cu arises from the copper grid used.

In this synthesis of Bi₂S₃ nanorods using microwave irradiation technique, starch has been utilized as the stabilizing agent because starch reduces the cytotoxicity of the nanoparticles, which arises from its biodegradable nature. In addition, starch has several advantages, such as inexpensive, hydrophilic, non-toxic, biocompatible, and abundantly available.²⁴ The formation mechanism of Bi₂S₃ nanorods using Bi(NO₃)₃ and Na₂S in an aqueous solution under microwave heating is proposed in Fig. 2. Under microwave irradiation, Bi³⁺ ions from Bi(NO₃)₃ react with S²⁻ ions from Na₂S to form Bi₂S₃ nuclei around the long chain of starch. These freshly formed Bi₂S₃ nuclei in the solution are affected and controlled by starch to grow along the [0 0 1] direction into Bi₂S₃ nanorods.^1,25,26

Fig. 2 Formation mechanism of the Bi₂S₃ nanorods.

3.2 Interaction mechanism between HSA and Bi₂S₃ nanorods

In spite of having three intrinsic fluorophores, such as tryptophan (Trp), tyrosine (Tyr) and phenyl alanine (Phe), in HSA, most of the fluorescence characteristics of HSA have been contributed by the Trp and Tyr residues because of the low fluorescence quantum efficiency of Phe. HSA has only one Trp residue, located at amino acid residue position 214 located in the sub-domain IIA, and 17 tyrosine residues.²⁷ Since the intrinsic fluorescence is very sensitive to environmental changes,²² the fluorescence experiments were carried out in the absence and the presence of Bi₂S₃ nanorods (Fig. 3a). The fluorescence emission spectrum of HSA shows a strong fluorescence emission peak at 335 nm with excitation at 280 nm. While increasing the concentration of Bi₂S₃ nanorods from 0 to 4 μM, the fluorescence intensity of HSA decreases gradually along with a blue shift in the emission maxima, which indicates that the Bi₂S₃ nanorods interact with HSA and quench its intrinsic fluorescence along with increasing hydrophobicity of the fluorophore microenvironment.^17,27

Fig. 3 Fluorescence spectra at λ_ex = 280 nm (a), fluorescence decay curves (b) and absorption spectra (c) of HSA (4 μM) quenched by Bi₂S₃ nanorods in the concentration range of 0–4 μM. The absorbance of Bi₂S₃ nanorods (4 μM) at 280 nm found from the absorption spectrum (c) is 0.0257.

The quenching mechanisms between the fluorophore and the quencher can usually be classified as either static or dynamic, in which static quenching is caused by the non-fluorescent ground state complex formation, whereas dynamic quenching results from the collisional encounters. The static quenching mechanism can be distinguished from the dynamic quenching by their differing dependence on temperature and viscosity, absorption spectroscopy or preferably by the fluorescence lifetime measurements.^17,18

While considering the fluorescence lifetimes, the quencher will shorten the fluorescent lifetime of the excited molecules in dynamic quenching, but for static quenching, the quencher does not affect the fluorescence lifetime of the molecule.^17,27,28 No remarkable change in the fluorescence lifetime decay of HSA was observed even upon the addition of Bi₂S₃ nanorods (Fig. 3b), which confirms that the static quenching mechanism occurs upon the interaction of HSA with Bi₂S₃ nanorods.

Absorption spectroscopy is a simple, versatile and effective method for confirming the possible quenching mechanism. Static quenching influences the absorption spectra of the fluorophore, whereas dynamic quenching influences only the fluorescence spectra.^17,29 In this regard, the absorption spectra of 4 μM HSA with varying Bi₂S₃ nanorods concentration range of 0–4 μM were measured (Fig. 3c). While adding Bi₂S₃ nanorods, the absorption intensity increases with a blue shift in the maximum absorption wavelength of HSA around 278 nm, which corresponds to the n → π* transition of the peptide bond as well as the amino acid residues such as Trp, Tyr, and Phe of HSA. This blue shift indicates the interaction of HSA with Bi₂S₃ nanorods through a static quenching process, i.e., ground state complex formation.^17,18,28,29 Therefore, the equilibrium for the interaction of HSA with Bi₂S₃ nanorods represented in Fig. 1a, in which K_app represents the apparent association constant. This K_app can be obtained from the absorption intensity changes of the peak maxima using the equation reported in the literature as follows:^20,30

where A_obs is the observed absorbance of the solution containing different concentrations of Bi₂S₃ nanorods at the peak maxima, A₀ is the absorbance of HSA, and A_comp is the absorbance of the complex at peak maxima. The value of the apparent association constant (K_app), determined from the linear relationship between 1/(A_obs − A₀) versus the reciprocal concentration of Bi₂S₃ nanorods (Fig. S1, ESI†) with a slope equal to 1/[K_app(A_comp − A₀)] and an intercept equal to 1/(A_comp − A₀), was found to be 6.21 × 10⁴ M⁻¹.

The Stern–Volmer equation²⁷ can also be used to recognize the quenching mechanism:

where F₀ and F are the fluorescence intensities of serum albumins in the absence and presence of quencher; τ₀ is the average lifetime of the fluorophore (HSA) in the absence of quencher; K_SV, K_q and [Q] are the Stern–Volmer quenching constants, bimolecular quenching rate constant (or the efficiency of quenching) and the quencher concentration, respectively. HSA titrated by Bi₂S₃ nanorods shows a linear Stern–Volmer plot (Fig. S2, ESI†) with correlation coefficient r = 0.9995. From the slope of the linear plot, the Stern–Volmer quenching constant K_SV was found to be 7.11 × 10⁴ M⁻¹. Since the lifetime of HSA, τ₀, was found to be 5.33 × 10⁻⁹ s from the fluorescence lifetime decay studies, the bimolecular quenching rate constant K_q for the interaction of HSA with Bi₂S₃ nanorods is 1.33 × 10¹³ M⁻¹ s⁻¹, which is greater than the maximum scattering energy transfer between various quenchers and biopolymers collisional quenching (2.0 × 10¹⁰ M⁻¹ s⁻¹). This again suggests that a static quenching mechanism was predominant in the interaction of HSA with Bi₂S₃ nanorods.

3.3 Binding constant and the binding site

The binding constant (K_A) of the Bi₂S₃ nanorods with HSA and the number of binding sites (n) can be found using the Hill equation as follows:from the intercept and the slope of the linear plot obtained in the double-logarithm regression curves of log[(F₀ − F)/F] versus log[Q] (Fig. S3, ESI†), the values of K_A and n were determined to be 3.55 × 10⁴ M⁻¹ and 0.9453. This value of n close to 1 indicates the presence of only one class of independent binding site in HSA for Bi₂S₃ nanorods, which suggests the possible formation of 1 : 1 complex between HSA and Bi₂S₃ nanorods.^31,32 This binding constant in the range of 1–15 × 10⁴ M⁻¹ suggests moderate binding between HSA and Bi₂S₃ nanorods, which can be attributed to the formation of a less stable HSA–Bi₂S₃ nanorods complex. This binding constant, K_A, was further used to calculate the free energy change (ΔG°) using the equation as follows:

ΔG° = −RT ln K_A (4)

where R is the gas constant (8.314 J mol⁻¹ K⁻¹) and T is the temperature (298 K). At 298 K, the standard free energy change ΔG° was estimated to be −2334 J mol⁻¹, which indicates that the interaction of HSA with Bi₂S₃ nanorods was spontaneous.²⁷

Synchronous fluorescence spectroscopy is a simple, selective and sensitive spectral method for providing information on the molecular microenvironment in the vicinity of the fluorophore functional groups by synchronously scanning the excitation and emission monochromators, while maintaining a constant wavelength interval (Δλ) between them. In HSA, the synchronous fluorescence will provide information characteristic of Trp and Tyr residues by noticing the shift in the emission maximum (λ_em) when Δλ is stabilized at 60 and 15 nm, respectively.^29,30 The effect of the Bi₂S₃ nanorods on the synchronous spectra of HSA showed that the successive addition of Bi₂S₃ nanorods gradually decreased the intensity along with the slight blue shift when Δλ is 60 nm (Fig. 4a), whereas no shift was observed when Δλ is 15 nm (Fig. 4b). This blue shift indicates the increase in the hydrophobicity of the microenvironment around the Trp residue upon the addition of Bi₂S₃ nanorods, resulting in an increase in the hydrophobicity of the fluorophore environment.³³

Fig. 4 Synchronous fluorescence spectra [(a) Δλ = 60 nm and (b) Δλ = 15 nm] of HSA (4 μM) quenched by Bi₂S₃ nanorods in the concentration of 0 and 4 μM.

3.4 Fluorescence resonance energy transfer

Förster theory of non-radiative energy transfer or fluorescence resonance energy transfer (FRET) can be used to calculate the distance ‘r₀’ between Bi₂S₃ nanorods and the Trp residue of HSA due to the good overlapping (Fig. S4, ESI†) between the absorption spectrum of the acceptor (Bi₂S₃ nanorods) and the fluorescence spectrum of donor (HSA).³⁴ The energy transfer is related not only to the distance ‘r₀’ between the acceptor and donor, but also to the critical energy-transfer distance ‘R₀’, i.e., the critical distance when the transfer efficiency is 50%. Therefore,where E is the energy transfer efficiency, which can be determined experimentally from the fluorescence spectrum of donor with (F) and without (F₀) the acceptor concentration using the equation as follows^17,27,28,34R₀ can be calculated using the equation as follows:

R₀⁶ = 8.8 × 10⁻²⁵k²N⁻⁴ϕ_DJ (7)

where k² is the spatial orientation factor related to the geometry of the donor and acceptor dipoles (2/3), N is the refractive index of the medium (1.336), ϕ_D is the fluorescence quantum yield of the donor (0.15) and J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor.^35–37 J can be calculated using the equation as follows:where F(λ) is the fluorescence intensity of the donor at wavelength range of λ–λ + Δλ and ε(λ) is the extinction coefficient of the acceptor at wavelength λ. Using eqn (5)–(8), the values of E, R₀ and r₀ has been found out to be 11.93%, 4.07 nm and 5.68 nm, respectively. The donor to acceptor distance, ‘r₀’, is less than 7 nm, which is within the range of 0.5R₀ to 1.5R₀, indicating the efficient energy transfer from HSA to Bi₂S₃ nanorods.^{17,28–30,34–37}

3.5 Circular dichroism

CD spectroscopy is a very sensitive analytical technique to examine the secondary and tertiary structure of proteins while interacting with different molecules. The far-UV CD spectrum of HSA (Fig. 5a) shows two negative peaks around 208 and 222 nm, which were attributed to the n → π* transition of the α-helical peptide bonds. A decrease in the intensity was observed upon the addition of Bi₂S₃ nanorods, which indicates a decrease in the α-helix of HSA. The CD results are expressed in terms of the mean residue ellipticity (MRE) in deg cm² dmol⁻¹ according to the equation as follows:where θ_obs is the observed ellipticity in millidegrees, C is the molar concentration of the HSA, n is the number of amino acid residues in HSA (585), and l is the path length in centimeters.^30,31 The α-helical content of HSA was calculated from the MRE value at 208 nm using the equation as follows:where MRE₂₀₈ is the observed MRE value at 208 nm, 4000 is the MRE of the β-form and random-coil conformation cross at 208 nm, and 33 000 is the MRE of a pure α helix at 208 nm. Using the equations, the α-helix content in the secondary structure of HSA was determined to be 61.73% and 59.83% in the absence and presence of Bi₂S₃ nanorods. This decrease in the α-helix percentage indicates that Bi₂S₃ nanorods bound to the amino acid residues of main polypeptide chain of HSA, which caused slight unfolding of the polypeptide, leading to the conformational changes in HSA.^17,29

Fig. 5 Far UV (a) and near UV (b) circular dichroism spectra of HSA (4 μM) in the absence and presence of Bi₂S₃ nanorods (4 μM).

The tertiary structural changes can be investigated using the near-UV CD spectra of HSA in the absence and presence of Bi₂S₃ nanorods (Fig. 5b). The near-UV CD spectra of HSA shows two negative bands at 263 and 268 nm as well as two positive peaks at 275 and 290 nm, which are characteristic of disulfide and the symmetric environment of aromatic amino acid residues. On the addition of Bi₂S₃ nanorods, the negative peaks at 262 and 268 nm as well as the positive peaks at 275 and 290 were altered significantly, which indicates the partial disruption in the tertiary structure of HSA. The CD-spectral analysis showed that the overall conformation of HSA was disrupted in the presence of Bi₂S₃ nanorods.^23,38,39 The difference ellipticity curve in Fig. 5b, which is the difference between the CD contribution of HSA in the presence and absence of Bi₂S₃ nanorods, displays negative extrema at 295 nm with less intense peaks between 270 and 290 nm. This again shows that Bi₂S₃ nanorods interact more with the tryptophan moiety than the tyrosine moiety.^40,41

4. Conclusions

A simple microwave irradiation method was employed to synthesize starch-stabilized Bi₂S₃ nanorods, which were then well characterized. XRD along with HRTEM, and SAED showed the formation of orthorhombic structured well crystalline Bi₂S₃ of ca. 15 nm in width with a rod-like morphology. The binding characteristics of the microwave synthesized Bi₂S₃ nanorods with HSA were investigated using multispectroscopy techniques. The collective UV-Vis and fluorescence (steady-state, time-resolved and synchronous) spectroscopy results determined that the Bi₂S₃ nanorods bind spontaneously to HSA affecting the tryptophan moiety through a static mechanism with a binding distance of 5.68 nm. The CD spectral result suggests that HSA retained quite a lot of the secondary structure as well as a significant proportion of the tertiary structure, while interacting with the Bi₂S₃ nanorods. Knowledge of this interaction provides valuable information that can be used for a better understanding of the design and applications of future nanomedical devices based on Bi₂S₃ nanorods.

Acknowledgements

The authors SN and RVM gratefully acknowledge the FONDECYT Post-doctoral Project No. 3150102 and FONDECYT No. 1130916, Government of Chile, Santiago, for the financial assistance. The authors SA and JJW thank the Department of Science and Technology, India (GITA/DST/TWN/P-50/2013) and the National Science Council (NSC), Taiwan (NSC-102-2923-035-001-MY3), respectively, for the India–Taiwan collaborative research grant.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra22641d

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