Studies on the Interaction of Imidazolium Ionic Liquids with Human Serum Albumin

Lavkesh Kumar Singh Tanwar¹, Kallol K Ghosh^2*

^1,2School of Studies in Chemistry, Pt. Ravishankar Shukla University, Raipur-492010(C.G.), India.

Abstract

Imidazolium-based ionic liquids have emerged as promising bio-compatible solvents for bio-molecules. The interaction of two imidazolium-based ionic liquids, namely 1-decyl-3-methyl-imidazolium tetrafluoroborate [Dmim][BF₄] and 1-butyl-3-methylimidazolium octylsulfate [Bmim][OS], with human serum albumin (HSA) have been investigated using UV-visible, fluorescence and fourier transform infrared spectroscopy. Stern-Volmer quenching constant (K_sv) and the binding affinity (K_a) value have been also calculated to reveals the molecular interactions between HSA and the imidazolium-based ILs. Additionally, we explored the thermodynamic feasibility of these interactions by calculating the Gibbs free energy (∆G), entropy (∆S), and enthalpy (∆H). Hydrophobic interactions have been identified as exerting a more significant influence than hydrogen bonding in the interactions between proteins and ionic liquids. This implies that the hydrophobic characteristics of the ionic liquids play a pivotal role in the denaturation of proteins. Consequently, we conclude that the hydrophobic nature of the ionic liquids is essential for inducing interactions with proteins and potentially contributing to protein structure denaturation.

Keywords: Ionic liquid, Imidazolium ionic liquids, Serum albumin, Fluorescence, FTIR.

1. Introduction

Serum albumin, the predominant protein in the circulatory system, plays a crucial role in various physiological processes, including the transportation and binding of compounds like fatty acids, drugs, and hormones  (Sindhu et al., 2022; Rawat and Bohidar, 2012; Egorova et al., 2017). The interaction of serum albumin with different molecules and solvents significantly affects its structure and function (Lie et al., 2017; Welton et al., 1999). Imidazolium-based ionic liquids (ILs) have gained attention due to their unique properties, with varying alkyl chain lengths (Reddy et al., 2023; Darlington et al., 2023; Fan et al., 2022; Singh et al., 2021). Short chain ILs impact protein conformation and enzymatic activity, while long chain ILs can induce denaturation (Wang et al., 2012; Das et al., 2014; Akdogan et al., 2011; Singh et al., 2018; Anand et al., 2011). Understanding their effects on serum albumin is vital for biomedical applications. This study aims to investigate these interactions, providing insights for drug delivery and protein stabilization.

Proteins' interaction with various compounds like ILs, drugs, and surfactants has been extensively studied, elucidating molecular interactions during protein denaturation. High IL concentrations can denature proteins like Human Serum Albumin (HSA). Studies by Shu et al. (2011) and Sindhu et al. (2020) demonstrated that imidazolium ILs quench BSA fluorescence and induce unfolding via hydrophobic and electrostatic interactions. Venkatesu et al. (2015) investigated how counter-ions in imidazolium ILs affect α-chymotrypsin. Imidazolium ILs can unfold HSA's tertiary structure via hydrogen bonding with amino acid residues. Shorter alkyl chain ILs decrease protein stability and enantio-selectivity. This study explores how alkyl chain length in imidazolium ILs influences HSA interactions. Khachatrian et al. (2023) examined how various imidazolium- and cholinium-based ionic liquids (ILs) affect bovine serum albumin (BSA). Using techniques like circular dichroism and fluorescence spectroscopy, they found that most ILs increased BSA's thermal stability except [OMIM][BF₄]. Shorter alkyl chain ILs promoted BSA aggregation, enhancing its thermal stability.

In the present investigation, we have studied the effect of two imidazolium-based ionic liquids: 1-decyl-3-methylimidazolium tetrafluoroborate [Dmim][BF₄] and 1-butyl-3-methylimidazolium octylsulfate [Bmim][OS] with human serum albumin (HSA). Analytical techniques including UV-visible, fluorescence and fourier transform infrared spectroscopy (FT-IR) were employed to study the effect of these imidazolium-based ILs. Stern-Volmer quenching constant (K_sv) and the binding affinity (K_a) value have been also calculated to reveals the molecular interactions between HSA and the imidazolium-based ILs. Additionally, we explored the thermodynamic feasibility of these interactions by calculating the Gibbs free energy (∆G), entropy (∆S), and enthalpy (∆H). Furthermore, using FT-IR spectroscopy, involvement of different functional groups during the interaction between HSA and imidazolium ILs has been also studied.

2. EXPERIMENTAL SECTION

2.1 Materials

Human serum albumin (HSA)(purity ≥ 99.5 %), imidazolium based ionic liquids i.e.,1-decy-3-methyl-imidazolium tetrafluoroborate [Dmim][BF₄] (purity ≥ 99.5 %), 1-butyl-3-methylimidazolium octylsulfate [Bmim][OS], sodium hydrogen phosphate (Na₂HPO₄) (purity ≥ 99.5 %) and potassium di hydrogen phosphate (KH₂PO₄) (purity ≥ 99.5 %), were purchased from Sigma Aldrich Pvt. Ltd. Bangalore, India and were used without further purification. All the experiments were carried out in double distilled water. Molecular structure of imidazolium based ILs i.e., [Dmim][BF₄], [Bmim][OS] and HSA shown in Scheme 1.

2.2 Methods

2.2.1 UV-visible spectroscopy

The absorption spectra were recorded at room temperature using a Varian Cary-50 (Agilent Technology), UV-visible spectrophotometer. The absorption spectra of HSA as well as HSA-IL system were measured in the wavelength range of 200-400 nm in 0.2 M phosphate buffer (pH 7.4) used as medium. The absorbance measurements were performed by concentration based manner of ILs i.e., [Dmim][BF₄], [Bmim][OS] and HSA  concentration was kept constant.

2.2.2 Fluorescence spectroscopy

The fluorescence quenching mechanism were investigated on a Cary Eclipse Fluorescence Spectrophotometer using a quartz cell with 1.0 cm path length in a thermostatically controlled cell holder at room temperature. The fluorescence spectra HSA recorded under the excitation of 330 nm and in the range of 250–500 nm. Both excitation and emission slit widths were kept fixed at 5 nm.

2.2.3 Fourier transform infrared spectroscopy (FT-IR)

Infrared spectroscopic investigation were carried out by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) model: Nicolet iS10, Thermo Fisher Scientific Instrument, Madison, USA). All spectral measurements were carried out by averaging 32 scans at 4 cm^-1 resolution over the range of 4000-500 cm^-1.

3.1 UV-visible measurements

The absorbance spectra of HSA in the absence and presence of imidazolium-based ILs with short and long alkyl chains depict in Fig. 1. The λ_max for HSA was found to be at 280 nm, which is due to the π-π* transition typical of the polypeptide chain as well as the n-π* transition of aromatic amino acids such as tyrosine (Tyrs), tryptophan (Tryp), and phenylalanine (Phen) (Jha et al., 2015).

The concentration of HSA remained constant throughout the study, while both imidazolium-based ILs, [Dmim][BF₄] and [Bmim][OS], were maintained at a concentration of 2 mM. The critical micelle concentration (CMC) of the ILs was determined using conductivity and surface tension measurements, as depicted in Fig. 1. The CMC values were found to be 1 mM for [Dmim][BF₄] and 30 mM for [Bmim][OS] (Hua et al., 2012; Geng et al.,2009). Upon the addition of [Dmim][BF₄] and [Bmim][OS], the absorption spectra of HSA exhibited a gradual increase. It has been previously reported that at higher IL concentrations, protein denaturation may occur (Sindhu et al., 2020). This denaturation can be inferred from the red shifts observed in the absorption spectra of HSA. These spectral changes are likely a result of structural modifications in the polypeptide chain caused by the binding of imidazolium-based ILs to HSA (Patel et al., 2014). Typically, the amide moieties in proteins that are exposed to a water environment undergo a low-energy π-π* transition in the UV region. In the excited state, the π* electron cloud exhibits higher polarity than the π electron cloud due to the formation of an antibonding orbital between carbon and oxygen atoms (Geng et al., 2010; Kelly et al., 2003).

The presence of ILs leads to a modification of the microenvironment surrounding the amide moieties in HSA. As the ILs displace the polar water molecules with their weaker polar nature, the energy required for the π* transition decreases, resulting in a bathochromic shift (Wang et al., 2012; Lim and Klahn, 2018). The addition of [Dmim][BF₄] and [Bmim][OS] ILs, considering the n-π* transition of aromatic amino acids, suggests a change in the microenvironment of these amino acids due to hydrophobic interactions with HSA (Umapathi et al., 2017). It is worth noting that the hydrophobic interaction has a significant influence on the microenvironment around the peptide chain compared to other interactions such as van der Waals and electrostatic interactions (Singh et al., 2012). The hydrophobicity of [Dmim][BF₄] and [Bmim][OS] differs due to the variation in alkyl side chain length in the cationic imidazolium moieties, which plays a major role in the interaction between the peptide chain of HSA and ILs (Shu et al., 2011). The hydrophobic interaction between [Dmim][BF₄] and the peptide chain is greater compared to [Bmim][OS]. The absorption spectra of the [Dmim][BF₄]-HSA and [Bmim][OS]-HSA systems exhibit a gradual increase with increasing IL concentration, indicating the binding of both imidazolium ILs to HSA. Additionally, a slight red shift in the absorption spectra is observed as the IL concentration increases, possibly indicating denaturation of HSA (Constatinescu et al., 2010; Herrmann et al., 2012). The binding affinity of both imidazolium-based ILs with HSA was also studied at three different temperatures, further emphasizing the crucial role of the alkyl chain length in the interaction between ionic liquids and proteins.

Fig. 1 UV-visible spectra of HSA with [Dmim][BF₄] and [Bmim][OS] at three temperature (A) HSA-[Dmim][BF₄] at 295 K, (B) HSA-[Bmim][OS] at 295 K, (C) HSA-[Dmim][BF₄] at 298 K, (D) HSA-[Bmim][OS] at 298 K, (E) HSA-[Dmim][BF₄] at 305 K and (F) HSA-[Bmim][OS] at 305 K.

3.1.1 Determination of binding constant

The binding affinity between HSA and [Dmim][BF4] as well as [Bmim][OS] was examined at three different temperatures. The binding constants for the HSA-[Dmim][BF4] and HSA-[Bmim][OS] systems were calculated from the UV-visible data using the Benesi-Hildebrand  Eq. (1) (Pal et al., 2016):

                                                             (1)

where, A₀, A and A_max are the absorbance in the absence and presence of [Dmim][BF₄] /[Bmim][OS] and maximum absorbance at saturation point, respectively. K_a is the binding constant. The calculated binding constants are tabulated in Table 1. These values were obtained from the slope of the plot of 1/[A-A₀] versus 1/[Q], as depicted in Fig. 2. The R² value of approximately 0.998 for all systems suggests the formation of a 1:1 complex between HSA and [Dmim][BF₄]/[Bmim][OS] ILs. The binding constant values were determined at three different temperatures: 295 K, 298 K, and 305 K.

It was observed that the binding constant values were lower at 305 K compared to the other temperatures, indicating the denaturation of proteins at higher temperatures (Singh et al., 2010). Moreover, the binding constant value was found to be higher for the HSA-[Dmim][BF₄] system as compared to the HSA-[Bmim][OS] system.

3.2 Fluorescence measurements

Fluorescence technique is a crucial method for studying molecular interactions, providing valuable information about the interaction between molecules. The fluorescence quenching mechanism has been extensively investigated to understand the molecular interactions of proteins, such as HSA and BSA, with other molecules (Modi et al., 2019; Guria et al., 2020). Fluorescence quenching occurs when a fluorophore interacts with quencher molecules, leading to a decrease in fluorescence intensity and a reduction in the fluorescence quantum yield. In this study, we focused on the quenching of fluorescence intensity in HSA caused by two imidazolium-based liquids, namely [Dmim][BF₄] and [Bmim][OS]. The fluorescence spectra of HSA in the presence of [Dmim][BF₄] and [Bmim][OS] are shown in Fig. 3. The fluorescence property of HSA arises from the presence of three monomeric amino acids: phenylalanine (Phen), tryptophan (Trp), and tyrosine (Tyr). Under excitation at 280 nm, the fluorescence spectra of HSA exhibit a single peak at 340 nm. The fluorescence of HSA is mainly attributed to the strong fluorescence emitted by the aromatic amino acids Trp and Tyr (Yan et al., 2012). These aromatic amino acids are the primary contributors to the fluorescence properties of HSA. The fluorescence spectra of HSA, excited at 280 nm, primarily reflect the characteristic structure of the polypeptide backbone of HSA (Chevrot et al., 2015). Consequently, any structural changes in HSA can significantly influence its fluorescence properties.

3.2.1 Fluorescence quenching mechanism of HSA

Previous studies have reported that imidazolium-based ILs exhibit negligible fluorescence efficiency and very low quantum yield (Banjare et al., 2019). In our investigation, we measured the fluorescence spectra of HSA while varying the concentration of two imidazolium-based ILs: [Dmim][BF₄] and [Bmim][OS]. The concentration of both ILs was varied from 0 to 2 mM, while the concentration of HSA remained constant. The fluorescence intensity of HSA was found to be quenched as the concentration of both ILs increased, with excitation at 280 nm, as shown in Fig. 3. Notably, the fluorescence intensity of HSA was significantly quenched in the presence of [Dmim][BF₄] compared to [Bmim][OS]. This observation can be attributed to the hydrophobic interaction between the alkyl chain of [Dmim][BF4] IL and the polypeptide backbone structure of HSA (Shao, 2013). In the case of the [Bmim][OS]-HSA system, the hydrophobic interaction is less significant due to the shorter length of the alkyl chain  (Hu et al., 2005). The increased hydrophobicity of longer alkyl chain ILs enhances their interactions with HSA. This can be attributed to the fact that the hydrophobic regions on the surface of HSA are more complementary to longer alkyl chains due to their increased size and surface area. As a result, longer alkyl chains in ILs can more effectively interact with these hydrophobic binding sites on HSA, leading to stronger binding affinity. Additionally, longer alkyl chains in ILs may also provide more opportunities for van der Waals interactions with HSA, further strengthening the binding affinity. These findings of fluorescence quenching align with the results obtained from UV-visible spectra. It can be concluded that the excitation at shorter wavelengths provides crucial information about the interaction between ILs and HSA.

3.2.2 Determination of Stern-Volmer and Binding constant

Fluorescence quenching can occur through two mechanisms: static quenching and dynamic quenching (Mondal et al., 2019). Dynamic quenching involves collisions between the fluorophore and quencher while in the excited state, whereas static quenching occurs through collisions in the ground state (Reddy et al., 2023). In our study, we observed that the fluorescence intensity of HSA gradually decreases with increasing temperature, indicating a static quenching mechanism (Weert and Stella 2011). This suggests that the quenching of fluorescence in the presence of [Dmim][BF₄] and [Bmim][OS] occurs predominantly through static quenching. To quantify the quenching process, the Stern-Volmer quenching constant was calculated for the HSA-[Dmim][BF₄] and HSA-[Bmim][OS] systems using the following equation Eq. (3).

                                             K_sv [Q]                                                             (3)

where, K_sv is Stern-Volmer quenching constant, [Q] is the concentration of quencher i.e., [Dmim][BF₄] and [Bmim][OS]  , F_o and F are the Fl intensities in the absence and presence of quencher, respectively. The Stern-Volmer quenching constant (K_sv) was determined by performing linear regression analysis on the plot of F_o/F against [Q] for the HSA-[Dmim][BF₄] and HSA-[Bmim][OS] systems at three different temperatures (as shown in Fig. 4). The calculated values of K_sv are tabulated in Table 1. It was observed that the K_sv value was higher for the [Dmim][BF₄]-HSA system, indicating a stronger interaction and increased hydrophobic interactions with HSA. At higher temperatures, the K_sv value may be higher due to protein denaturation and changes in the secondary structure of HSA in the presence of imidazolium-based ILs (Halder et al., 2021). These results suggest that the quenching mechanism in both the HSA-[Dmim][BF₄] and HSA-[Bmim][OS] systems is predominantly static quenching. The Stern-Volmer quenching constant provides valuable information about the efficiency of the quenching process and the strength of the interaction between HSA and the ILs.

                                      The binding affinity of [Dmim][BF₄] and [Bmim][OS] with the HSA molecule was also examined using fluorescence measurements. The binding constant for the HSA-[Dmim][BF₄] and HSA-[Bmim][OS] systems was determined using a modified form of the Benesi-Hildebrand equation (Eq. 4) (Baker et al., 2019) This equation allows us to quantitatively analyze the binding interactions between the imidazolium-based ILs and HSA.                                               

                                                                          (4)

where, F₀ and F is the fluorescence intensity of pure HSA and intensity with addition of [Dmim][BF₄] and [Bmim][OS] respectively, F_max is the maximum fluorescence intensity, [Q] is the concentration of [Dmim][BF₄] and [Bmim][OS] and K_a is the binding constant. The plot of 1/(F₀ - F) versus 1/[Q] was constructed as shown in Fig. 4. The slope of this plot was used to calculate the binding constant (K_a) for the HSA-[Dmim][BF₄] and HSA-[Bmim][OS] systems at different temperatures. The calculated K_a values are presented in Table 1. It was observed that the K_a value was higher for the HSA-[Dmim][BF₄] system compared to the HSA-[Bmim][OS] system. This difference can be attributed to the stronger hydrophobic interactions between the longer alkyl chain of [Dmim][BF₄] and HSA (Chakraborty et al., 2009). The obtained K_a values are in good agreement with the results obtained from the UV-visible spectroscopy analysis.

Fig. 3 Fluorescence spectra of HSA with [Dmim][BF₄] and [Bmim][OS] at three temperature (A) HSA-[Dmim][BF₄] at 295 K, (B) HSA-[Bmim][OS] at 295 K, (C) HSA-[Dmim][BF₄] at 298 K, (D) HSA-[Bmim][OS] at 298 K, (E) HSA-[Dmim][BF₄] at 305 K and (F) HSA-[Bmim][OS] at 305 K.

Fig. 4 Stern-Volmer plots of fluorescence quenching of HSA by [A] [Dmim][BF₄] and [B] [Bmim][OS] at three temperature and Plots of log log[F⁰-F/F] vs. log [Q] for binding constant of HSA λmax = 280 nm at three temperature (pH 7.6) i.e., (C) HSA-[Dmim][BF₄] and (D) HSA-[Bmim][OS].

Table. 1 Binding constant and Stern-Volmer constant values for HSA-[Dmim][BF₄] and HSA-[Bmim][OS] at 295 K, 298 K and 305 K.

3.2.3 Determination of thermodynamic parameter

To evaluate the spontaneity of the interaction between HSA and [Dmim][BF₄] and [Bmim][OS], various thermodynamic parameters including Gibbs free energy (∆G), enthalpy (ΔH), and entropy (ΔS) were determined using the van 't Hoff equation at three distinct temperatures: 295 K, 298 K, and 305 K. The binding constant values obtained from the Benesi-Hildebrand equation were utilized to calculate the Gibbs free energy using the following equation (2):

                                                     ΔG = ─RTlnK_a                                                                                                  (2)

where, R is the gas constant (8.314 J mol^-1 K^-1), T is the temperature and K_a is the binding constant. The calculated values of Gibbs free energy have been tabulated in Table 2. It is observed that the value of Gibbs free energy found to be negative for both HSA-[Dmim][BF₄] and HAS-[Bmim][OS] system which indicates the thermodynamic feasibility of interaction at three different temperatures.

Table 2 Thermodynamic parameters, such as, Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) for the HSA-[Dmim][BF₄] and HSA-[Bmim][OS] at 295 K, 298 K and 305 K.

The negative value of ΔG indicates that the binding process between HSA and ILs is spontaneous. Similarly, the negative value of ΔH suggests that heat is released during this binding interaction. The change in entropy (ΔS) plays a crucial role in determining the driving forces associated with the HSA-ILs interaction. The entropy value also confirms the feasibility of the binding process for both ILs. Furthermore, the negative ΔH value indicates that the reaction is exothermic. The obtained values of various thermodynamic parameters clearly demonstrate that the binding of HSA with [Dmim][BF₄] and [Bmim][OS] is thermodynamically favourable.

3.3 FT-IR study

The FT-IR study have been applied to reveals the functional groups interaction between imidazolium ILs and HSA. The FT-IR spectra of pure imidazolium based ILs i.e., [Dmim][BF₄] and [Bmim][OS] and their mixture with HSA have been  shown in Fig. 5 and their stretching frequencies tabulated in Table 3. It has been observed that the asymmetric and symmetric vibration of –CH­₂ of imidazolium ILs participated during the interaction. The stretching frequency of imidazolium ILs shifted in presence of HSA indicates the strong interaction take place which is driven by various interaction forces such electrostatic and van dar Waals interaction.  Banjare et al., (2019) studied the interaction of [Bmim][OS] with globular proteins using FT-IR spectroscopic techniques, and they observed that the stretching frequency of amide group of globular protein shifted in presence of [Bmim][OS] due to rearrangement of secondary structure of serum albumin.

3.3.1 FT-IR study of 1-decyl-3-methyl imidazolium-tetrafluoroborate ionic liquid

The FTIR spectra of [Dmim][BF₄]  shown in Fig. 5 (A). It can be observed that the peak at 2901.22 cm^-1 is for C-H stretching, stretching frequency of ─CH₂ observed at 1472.31 cm^-1, for C-H of CH₃-N⁺ peak observed at 1541.01 cm^-1, C-H in plane-bending is 1121.10 cm^-1, aromatic C-H bending is 737.12 cm^-1 for [Dmim][BF₄] respectively.

3.3.2 FT-IR study of 1-butyl-3-methyl imidazolium-octylsulphate ionic liquid

The FT-IR spectra of [Bmim][OS]  shown in Fig. 5 (B). It can be observed that the peak at 2958.64 cm^-1 is for symmetric and asymmetric stretching of –CH₂, frequency observed at 1566.75 and 1460.36 is for symmetric and asymmetric stretching for C-H of CH₃-N⁺ moiety. The vibration frequency observed at 749.98 is attributed to S-O symmetric stretching for [Bmim][OS] respectively.

3.3.3 Interaction of ionic liquid with HSA studied by FT-IR

The interaction of different functional between HSA and imidazolium based ionic liquids has been revealed by the FT-IR measurements. The FT-IR spectra of HSA-IL complex shown in Fig. 6 (A) and (B). The [Dmim][BF₄]-HSA  complex shown in Fig. 6 (A).  It is observed that the peak of C-H stretching 2901.22 cm^-1 is shifted to 2921.06 cm^-1, stretching frequency of [CH₂] 1093.21 cm^-1 is shifted to 1057.30 cm^-1, stretching frequency of aromatic C=C bending shifted to 1562.77 cm^-1, aromatic C-H bending shifted to 740.83 cm^-1 from 737.12 cm^-1.

Table. 3 FTIR data for the pure imidazolium ILs i.e., [Dmim][BF₄] and Bmim][OS] and their complex with HSA [Dmim][BF₄]-HSA and [Bmim][OS]-HSA.

The FT-IR spectra of HSA-[Bmim][OS] complex shown in Fig. 6 (B). It can be observed that the symmetric and asymmetric stretching of –CH₂ is shifted to peak at 2872.85 cm^-1. The symmetric and asymmetric stretching for C-H of CH₃-N⁺ moiety is shifted to 1166.72 cm^-1 and 1565.72 cm^-1. The shifting asymmetric and symmetric vibration of –CH­₂ of imidazolium based ILs confirms participated of ILs during the interaction.

In this study, we have investigated the effect of two imidazolium-based ionic liquids, i.e., 1-decyl-3-methyl-imidazolium tetrafluoroborate [Dmim][BF₄] and 1-butyl-3-methylimidazolium octylsulfate [Bmim][OS], on human serum albumin. UV-visible, fluorescence, and Fourier transform infrared spectroscopic techniques were employed to explore molecular interactions. Our findings revealed that ionic liquids with longer alkyl chains, such as [Dmim][BF₄], displayed higher binding affinity towards HSA, inducing notable conformational alterations in the protein. This phenomenon can be attributed to the larger size and surface area of longer alkyl chains, which better complement the hydrophobic regions on the surface of HSA. Consequently, longer alkyl chains in ILs can more efficiently engage with these hydrophobic binding sites on HSA, resulting in enhanced binding affinity.   Furthermore, we have calculated the thermodynamic parameters, indicating that the interactions between the protein and ionic liquids occurred spontaneously. Hydrophobic interactions played a significant role in mediating these interactions, surpassing the contribution of hydrogen bonding. These findings provide valuable insights into the toxicity profile of ionic liquids with long alkyl chains and enhance our understanding of the mechanisms underlying their interaction with HSA across various concentration ranges.

 Acknowledgments                                                                                               

Lavkesh Kumar Singh Tanwar gratefully acknowledges fellowship received from the Pt. Ravishankar Shukla University, Raipur (C.G.). The authors are grateful to the National Center for Natural Resources, Pt. Ravishankar Shukla University, Raipur (C.G.) for providing the FTIR analysis.

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