Bipyridinium and Imidazolium Ionic Liquids for ... - ACS Publications

Research Article pubs.acs.org/journal/ascecg

Bipyridinium and Imidazolium Ionic Liquids for Nanomaterials Synthesis: pH Effect, Phase Transfer Behavior, and Protein Extraction Rajni Aggarwal,§ Poonam Khullar,*,§ Divya Mandial,§ Aabroo Mahal,§ Gurinder Kaur Ahluwalia,‡ and Mandeep Singh Bakshi*,† †

Department of Natural and Applied Sciences, University of Wisconsin−Green Bay, 2420 Nicolet Drive, Green Bay, Wisconsin 54311-7001, United States ‡ Nanotechnology Research Laboratory, College of North Atlantic, Labrador City, NL A2V 2K7 Canada § Department of Chemistry, B.B.K. D.A.V. College for Women, Amritsar 143005, Punjab, India S Supporting Information *

ABSTRACT: We demonstrate the potential use of 1,1′-bis(2(cyclohexyloxy)-2-oxoethyl)-[4,4′-bipyridin]-1,1′-diium bromide (BP) and 1-ethyl-3-methylimidazolium chloride (EMI) ionic liquids (ILs) in in situ synthesis of gold nanoparticles (Au NPs) without using any external reducing or stabilizing agents. Both ILs produced nearly monodisperse NPs of 4−8 nm which were present in the form of self-assembled states. BP coated NPs formed self-assembled sheets and easily transferred to the organic phase by employing the water insoluble IL as a phase transfer agent. The efficiency of the phase transfer process was related to the extent of aggregation as well as functional groups. Both IL coated NPs were further used to extract the proteins from the complex biological mixtures. EMI coated NPs extracted proteins of large molar masses whereas BP coated NPs were good for the extraction of low molecular mass proteins. This disparity was controlled by the substituted functional groups of ILs. Bulky cyclohexyloxy functional groups of BP did not allow extraction of large molar mass proteins. Such a wide applicability of ILs in nanomaterials synthesis opens several new applications in the field of nanomedicine and nanobiotechnology where IL coated NPs can be used for diverse protein complexation. KEYWORDS: Ionic liquid coated nanoparticles, Phase transfer, Hemolysis, Protein extraction

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INTRODUCTION Ionic liquids (ILs) belong to a unique category of unconventional liquids with enormous applications in green and environmental chemistry.1,2 They are the versatile solvents for various organic, inorganic, and polymeric molecules and provide suitable solution behavior over a wide range of temperatures from subzero to as high as of 200 °C.3−5 They are of low volatility, inexpensive, and easy to synthesize. Though, they are widely used to enhance nanoparticle (NP) stability, physiochemical behavior, and catalytic properties,6−8 the evaluation of their potential in NP synthesis as stabilizing and reducing agents requires a systematic investigation. They are ionic and, hence, have inherent ability for surface adsorption and complexation on metal NPs to develop an electric double layer that in turn provides charge stabilization and generates colloidal stability, essential for shape controlled crystal growth as well as the photophysical properties of NPs.9,10 However, apart from the electrostatic interactions, other forces such as van der Waals interactions and H-bonding also play a significant role when functionalized ILs are used. Such ILs induce a high degree of self-organization of IL coated NPs in the liquid state.11,12 This kind of network liquid-phase structure © 2017 American Chemical Society

can be used as a template for a well-defined nanoscale architectures with long-range order and can influence the synthesis, stability, and growth of nanomaterials. We demonstrate this by choosing two ionic liquids with contrasting different characteristic features in the synthesis of nanomaterials. One is bipyridinium bromide [1,1′-bis(2-(cyclohexyloxy)-2oxoethyl)-[4,4′-bipyridin]-1,1′-diium bromide (BP)] and the other is imidazolium chloride [1-ethyl-3-methylimidazolium chloride (EMI)] (Figure 1). Both ILs differ in their functionalities. BP possesses cationic bipyridinium and cyclohexyloxy functional groups while EMI represents imidazolium. We demonstrate that how different functional groups ILs can be applied for different applications, and it has been shown by using them in nanomaterials synthesis, phase transfer behavior, and protein extraction. All these properties are highly dependent on the nature of the functional groups and have been shown by comparing the behaviors of BP and EMI ILs. Therefore, both ILs have been used for the in−situ synthesis of Received: May 1, 2017 Revised: July 16, 2017 Published: July 21, 2017 7859

DOI: 10.1021/acssuschemeng.7b01368 ACS Sustainable Chem. Eng. 2017, 5, 7859−7870

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ACS Sustainable Chemistry & Engineering

mass 21 kDa) was obtained from Sigma-Aldrich, rice protein was a gift from Dr. Narpinder Singh, GNDU, Amritsar, India, and blood serum was generated from the blood of a healthy human donor. In-situ Synthesis of Au NPs Using ILs. Both BP and EMI ILs have been used in in situ reaction conditions to synthesize Au NPs. A 10 mL portion of aqueous gold chloride solution (0.25−1 mM) and ILs (4−40 mM) were taken in a reaction bottle. After mixing the components at room temperature, the reaction mixture was kept in a water thermostated bath (Julabo F 25) at precisely 70 °C for 6 h under static conditions. Similar reactions were also conducted at pH = 12.5 to determine the influence of the basic medium on the synthesis of Au NP. The color of the solution changed from colorless to dark orange in all reactions where BP was used as a reducing as well as stabilizing agent, while dark rust colored NPs were obtained in all samples of EMI. NPs were collected by centrifuging the samples at 8000−12 000 rpm depending on their sizes and purified with distilled water at least 2−3 times. These reactions were also simultaneously monitored with the help of UV−visible (Shimadzu-Model No. 2450, double beam) and steady state fluorescence spectroscopy (PTI QuantaMaster) measurements. Both instruments were equipped with a TCC 240A thermoelectrically temperature controlled cell holder that allowed measurement of the spectrum at a constant temperature within ±1 °C. NPs were characterized by transmission electron microscopic (TEM) analysis on a JEOL 2010F at an operating voltage of 200 kV. The samples were prepared by mounting a drop of a solution on a carbon coated Cu grid and allowed to dry in the air. Hemolysis and Protein Extraction. Hemolytic Assay. Hemolytic assay was performed to evaluate the response of IL coated NPs on blood group B of red blood cells (RBCs) from a healthy human donor. Briefly, 5% suspension of RBCs was used for this purpose after giving three washings along with three concentrations (i.e., 25, 50, and 100 μg/mL) of each NPs sample. A 1 mL packed cell volume (i.e., hematocrit) was suspended in 20 mL of 0.01 M phosphate buffered saline (PBS). The positive control was RBCs in water and it was prepared by spinning 4 mL of 5% RBCs suspension in PBS. PBS as supernatant was discarded, and the pellet was resuspended in 4 mL of water. The negative control was PBS. All the readings were taken at 540 nm, i.e., the absorption maxima of hemoglobin. SDS Page Gel Electrophoresis. Protein extraction using IL coated NPs was carried out with the help of SDS Page analysis. Purified NPs were complexed with appropriate amounts of zein, rice protein, and serum aqueous solutions to extract the protein fractions. The protein complexed NPs were then purified twice with double distilled water and were used for SDS page analysis. Before the analysis, each purified sample was assorted with 1× sample loading buffer and boiled in a water bath at 100 °C to remove the protein corona adsorbed on the NPs surface. The suspension thus obtained is further centrifuged to remove NPs and supernatant containing adsorbed protein is subjected to SDS Page analysis. A 10 μL portion of the sample was loaded in the well of 5% stacking gel which was solidified over 12% separating gel. The loaded gel was immersed with 1× Tris-Glycine SDS gel running buffer and was electrophoresed at 120 V and 10 mA. The gel was stained and destained to obtain clear visible bands.

Figure 1. Molecular structures of different ionic liquids.

gold (Au) NPs under the effect of pH without using any external stabilizing or reducing agents to demonstrate their applicability in nanomaterials synthesis. Because of their charge stabilization and functionalities, IL coated NPs can be easily extracted into the organic phase from the aqueous phase in a simple phase transfer process by applying any other water insoluble IL such as 1-butyl-3-methyl imidazolium hexafluorophosphate (BMI) (Figure 1).13−15 This allows the applicability of functionalized IL coated NPs in industrial important nonaqueous phases such as paints, pigments, dyes, as well as in waste recycling of plastics and metal particulates. We demonstrate the applicability of such functionalized IL coated NPs in selectively extracting proteins from the complex biological mixtures such as unpurified protein suspensions and blood serum. This selective extraction of protein fractions provides plenty of opportunities for the applications of such nanomaterials in bionanotechnology.

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RESULTS AND DISCUSSION pH Effect on the Solution Behavior of BP. The aqueous solution of BP shows a dramatic color change under the effect of pH variation when successive amounts of aqueous NaOH are added. An increase in the pH makes the aqueous BP florescent and the emission intensity shows a dramatic variation (Figure 2a) with a color change from transparent to dark brown (Figure 2b). A plot of fluorescence intensity with pH indicates that it initially increases with pH up to 12.3 where the color of the solution turns light brown but thereafter, it decreases as the color intensifies with the further increase in pH (Figure 2c). Likewise, the wavelength undergoes a blue shift up to pH 12.3 and then shows a significant red shift. In basic medium, it is caused by the association of electron donating OH− ions to

EXPERIMENTAL SECTION

Materials. 1,1′-bis(2-(Cyclohexyloxy)-2-oxoethyl)-[4,4′-bipyridin]1,1′-diium bromide (BP) was synthesized as mentioned elsewhere16 while 1-ethyl-3-methylimidazolium chloride (EMI) and 1-butyl-3methyl imidazolium hexafluorophosphate (BMI) (Figure 1) were procured from Sigma-Aldrich. Chloroauric acid (HAuCl4), sodium citrate, sodium borohydride, cetyltrimethylammonium bromide (CTAB), and sodium dodecyl sulfate (SDS) were also purchased from Sigma-Aldrich and used for the in-situ as well as for the presynthesized Au NPs. Double distilled water was used for all preparations. For the protein extraction studies, zein protein (molar 7860


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Figure 2. (a) Fluorescence titration of aqueous solution of 10 mM BP with NaOH. (b) Corresponding color change of the solution at different pH. (c) Plots of fluorescence intensity and wavelength versus pH of the solution. (d) Similar titration by using UV−visible measurements, and (e) the variation of UV−visible intensity and wavelength with respect to pH. See details in the text.

bipyridinium centers that makes BP molecules fluorescent due to intramolecular charge transfer transitions.17 However, at higher pH, intermolecular association18 converts the radiative decay into nonradiative decay due to intermolecular collisions that reduce the intensity. Such color transformation produces two absorption bands in the visible region at 400 and 600 nm corresponding to the formation of two species (Figure 2d). The first one around pH 12.3 is the fluorescent molecule while its self-association produces another one around pH 12.6 which gives dark brown color (Figure 2e). No isosbestic point is observed in Figure 2d which demonstrates that both species exist independently, survive in the solution though the first one is fluorescent while the second is not, and, hence, produce two different absorbances in the visible region. EMI does not show this fluorescence or UV−visible behavior when titrated with NaOH (Supporting Information, Figure S1). In-situ Synthesis of Au NPs in the Presence of BP. When BP ionic liquid is used in the aqueous solution in a

neutral medium along with the gold salt for the synthesis of Au NPs (see the Experimental Section) and in the absence of any reducing or stabilizing agent, a dark orange suspension of Au NPs is obtained (Figure 3a, inset). TEM analysis shows the presence of sheets of BP wrapping tiny Au NPs (Figure 3a,b). High resolution image indicates the size of the Au NPs is 4−8 nm with clear crystal planes (Figure 3c). Formation of such small Au NPs suggests that BP is a fine reducing, stabilizing, as well as shape controlling agent. Formation of nearly monodisperse small spherical Au NPs is achieved because of the precise shape controlling effects of BP during the nucleation. Cationic bipyridinium ion of BP adsorbs preferentially on the {111} crystal planes and hence, passivate them from further participating in the nucleation while directing the growth at {110} and {100} crystal planes.10 That in turn leads to the formation of spherical morphologies. Such kind of remarkable shape control behavior is usually provided by the ionic surfactants. The origin of this is mainly attributed to the 7861


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Figure 3. (a) TEM image of BP coated Au NPs exist in the form of folded sheets. (inset) Sample prepared through an in-situ reaction using 10 mM of BP and 0.5 mM of gold chloride at 70 °C. (b) Single rolled sheet and (c) high resolution image of the crystal planes tiny Au NPs. (d and e) Phase transfer of BP coated Au NPs from aqueous to organic phase using BMI water insoluble IL. (f) UV−visible absorbance of Au NPs suspension. See details in the text.

self-assembled behavior19,20 of BP coated Au NPs which is driven by the van der Waals interactions between the nonpolar aromatic and cyclic functional groups. Such a self-assembled state of BP does not allow the anisotropic growth to happen and hence, is the consequence of the formation of fine nanomorphologies. Interestingly, such self-assembled NPs can be easily extracted into the organic phase by using appropriate water insoluble IL as a phase transfer agent. BMI is well-known water insoluble IL commonly used as a phase transfer agent. When a ethyl acetate solution of BMI is mixed with the aqueous suspension of BP stabilized Au NPs, NPs demonstrate a clear phase transfer from the aqueous to organic phase

(Figure 3d) driven by the electrostatic interactions between the oppositely charged ions of BP in the aqueous phase and that of BMI in the organic phase.13−15 Cationic bipyridinium coated Au NPs are electrostatically attracted by the anionic hexafluorophosphate in the organic phase and hence, the charge neutralization on Au NPs surface allows them to transfer into the organic layer. When the same reaction is conducted at pH = 12.5, a much higher yield of the Au NPs is obtained in comparison to that in the neutral medium. The final color of the Au NPs suspension is black (Figure 3e) rather than orange (Figure 3d) because of the greater yield in the former case that reduces the surface adsorbed amount of BP. At pH 12.5, 7862


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Figure 4. (a) TEM image of EMI coated Au NPs in the form of chainlike structures. (inset) Sample prepared through an in-situ reaction using 10 mM of EMI and 0.5 mM of gold chloride at 70 °C. (b and c) High resolution images and (d) phase transfer experiment where no phase transfer occurred. See details in the text.

bipyridinium functional groups of BP are neutralized by OH− groups, thereby rendering them predominantly nonpolar moieties with relatively less surface adsorption on NPs surface in comparison to that in neutral medium. However, these NPs too can be easily transferred to the organic layer by using BMI in the ethyl acetate medium (Figure 3e) and provide a prominent characteristic broad absorbance in the form of a plateau around 560 nm21,22 (Figure 3f). A broad absorbance is the consequence of several factors such as size, shape, and aggregation, and it is very common in the case of plasmonic NPs.10 Monodisperse Au NPs provide clear sharp absorbance around 520 nm when they exist in nonaggregated colloidal suspension; however, this absorbance becomes broad and red shifts10 when they exist in the aggregated state as in the present case. In-situ Synthesis of Au NPs in the Presence of EMI. For the sake of comparison, the above-mentioned reactions were carried out in well-known water-soluble ionic liquid EMI under similar reaction conditions.6,23 All Au NPs are settled at the bottom of the bottle as black color dust (see Figure 4a, inset) rather than an orange colloidal suspension observed in the presence of BP (Figure 3a, inset). The latter is the manifestation of cyclohexyloxy functional groups in BP that promote the nonpolar interactions in aqueous phase and hence, induce greater amount of self-aggregation among BP coated NPs which is not the case with EMI. TEM images of Au NPs thus obtained in neutral medium are shown in Figure 4a,b where EMI coated NPs exist in the form of chain like arrangements (Figure 4a,b) rather than self-assembled sheet

structures (Figure 3a,b). High resolution image (Figure 4c) indicates that the overall morphologies of NPs are similar to that of Figure 3c indicating a similar kind of crystal growth in both cases. However, the most contrasting difference among BP coated and EMI coated NPs is that the latter NPs do not transfer to the organic phase by using BMI under identical phase transfer conditions (Figure 4d) which is obviously due to a marked difference in the monovalent and divalent natures of cationic moieties of BP and EMI, respectively, as well as the presence of cyclohexyloxy functional group in BP (Figure 1). BP with divalent cationic (bipyridinium) moiety is expected to have stronger interactions with the hexafluorophosphate anion of BMI in comparison to that of monovalent cationic (1-ethyl3-methylimidazolium) moiety of EMI. Apart from this, a much greater amount of the BP coating in the form of self-assembled sheets makes it easier for the NPs to be easily transferred to the organic phase. In addition, we do not see any visible effect on the yield of the Au NPs when the same reaction in the presence of EMI is conducted at pH 12.5 in contrast to that of Figure 3e where a dramatic increase in the yield of Au NPs is observed. Hence, these results clearly demonstrate the contrasting difference in the surface adsorption behaviors between BP and EMI ions on the Au NPs that in turn determines the selfaggregation behaviors of Au NPs and their phase transfer from the aqueous to organic phase. Thus, the self-aggregation is promoted for BP coated NPs mainly because of the presence of the cyclohexyloxy moieties and that in turn facilities their phase transfer to organic phase in comparison to EMI coated NPs. These results are further evaluated by taking the presynthesized 7863


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Figure 5. (a) UV−visible titration of citrate stabilized Au NPs with BP. (b) Corresponding plots of intensity and wavelength versus the concentration of BP. (c) Similar titration of citrate stabilized Au NPs with EMI. (d) Photos showing how the titration in part a results in settled NPs can be redispersed into a colloidal suspension using NaOH or SDS. (e) Simultaneous monitoring using UV−visible measurements. (f) Corresponding intensity and wavelength plots. See details in the text.

Au NPs, i.e., Au NPs synthesized in the absence of any ionic liquids and then treated with both ionic liquids to understand their surface adsorption behaviors. Mode of Surface Adsorption of ILs Using Presynthesized Au NPs. In order to understand the mechanism of surface adsorption of ILs on the charged NPs surface, we have used presynthesized Au NPs. They were produced by mixing [HAuCl4] = 0.5 mM and sodium citrate [Na3Cit] = 0.5 mM, followed by the addition of 0.6 mL of ice cold aqueous NaBH4 ([NaBH4] = 0.1 mol/dm3) solution under constant stirring.21,22 This reaction provides negatively charged citrate stabilized Au NPs. However, if similar reaction is carried out in the presence of CTAB instead of Na3Cit, positively charged Au NPs of almost similar size are obtained. Both citrate and CTAB stabilized NPs suspensions provide equal colloidal stabilization at room temperature of polyhedral Au NPs of around 10 nm and are the best models to compare the surface adsorption of above-mentioned ILs. The surface adsorption is carried out by titrating the citrate stabilized NPs with BP and is simultaneously monitored with UV−visible measurements at 25 °C.

Such a titration is shown in Figure 5a where absorbance of Au NPs around 520 nm is monitored by adding successive amounts of BP. It leads to a dramatic color change of citrate stabilized Au NPs suspension from pink to dark purple (inset, Figure 5a). A variation in the intensity as well as wavelength versus the amount of BP is shown in Figure 5b where the intensity first increases with the increase in the amount of BP and then passes through a maximum with a steep fall. This causes a sharp increase in the wavelength with breakpoint corresponding to the maximum of the intensity curve. Initial rise in the intensity as well as wavelength is due to an instant deposition of the bipyridinium ions on negatively charged citrate stabilized surface of Au NPs to produce a stable electrical double layer that in turn provides greater colloidal stability to Au NPs.10 However, as more and more BP is deposited on the NPs surface, it screens the electric double layer due to the predominantly nonpolar nature of cyclohexyloxy functional groups and thus, induces a self-aggregation among the NPs in a much similar manner to that of Figure 3a. The color contrast between the orange suspension of Figure 3a and purple 7864


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Figure 6. (a) Typical UV−visible profiles of heme absorption of BP coated Au NPs of Figure 3a with positive control, negative control, 25, 50, and 100 μg/mL. (b) Corresponding representative photos of these samples. (c) Plots of hemolysis % versus dose of BP coated NPs (25, 50, and 100 μg/ mL) and (d) that of EMI coated NPs. Samples B1, B2, and B3 are prepared with 10 mM BP and 0.25, 0.5, and 1 mM gold chloride, respectively, while B4 is prepared with 20 mM BP and 0.25 mM gold chloride. The same respective compositions are used for E1, E2, E3, and E4 samples consisting of EMI coated Au NPs. See details in the text.

NPs surface as depicted in Figure 4 in the dried state, they lack the tendency of self-assembled behavior in the solution phase because of the absence of any predominantly hydrophobic functional group just like that of cyclohexyloxy functional groups of BP. Thus, self-assembled behavior of Au NPs (Figure 3a) as well as the phase transfer properties (Figure 3d,e) are only associated with BP coated Au NPs rather than the EMI cation coated NPs and are attributed to the presence of cyclohexyloxy functional groups in BP. Hemolysis. Hemolytic assay helps us to understand the cytotoxicity of IL coated NPs toward blood cells.24 Since ILs are charged and hence, they possess high affinity to interact with the blood cells membrane, and induce hemolysis. Hemolysis occurs when free NPs surface (uncoated) interacts with the various components of blood cell membrane through various forms of interactions viz. electrostatic, nonelectrostatic, and hydrogen bonding.25,26 Coated NPs usually do not induce hemolysis because complete passivation of the NPs surface that reduces the nanometallic surface to interact with the blood cell membranes.25,27 However, if coated molecules have the ability to disrupt the blood cell membrane, hemolysis cannot be avoided. That is why conventional ionic surfactant coated NPs have high potential to induce significant hemolysis.27 Similarly, ILs are charged like ionic surfactants and hence, interacts with the blood cell membrane which consists of three layers with glycocalyx on the exterior, protein network on the anterior, and lipid bilayer in between the two. Both BP and EMI coated NPs are subjected to hemolysis. Figure 6a demonstrates some of the representative UV−visible plots of heme absorption within

suspension of Figure 5a is simply due to the difference in the concentration of BP used, which is much higher i.e. Ten mM in the former in comparison to 50 μM of the latter. When the same titration is carried out by taking positively charged CTAB stabilized Au NPs (Figure 5c) instead of negatively charged citrate stabilized Au NPs, no marked change in the intensity or the wavelength of the absorbance of Au NPs is observed due to little interactions between the cationic bipyridinium ions and electropositive Au NPs surface. This confirms the surface adsorption of BP on NPs surface and is only driven by the cationic bipyridinium ions that control the crystal growth of the Au nucleating centers. We further show that the adsorption of bipyridinium ions on Au NPs surface is purely electrostatic in origin by treating the settled NPs of titration of Figure 5a by NaOH and SDS. It is illustrated in Figure 5d where settled NPs when titrated with NaOH, a stable colloidal suspension is regenerated. Simultaneous UV−visible measurements (Figure 5e) show an emergence of a prominent Au NPs absorbance around 700 nm from the initial broad absorbance of aggregated NPs around 750 nm. It increases and blue shifts (Figure 5f) as self-assembled arrangement of Au NPs dismantles by the successive additions of NaOH due to the stronger interactions of OH− ions with bipyridinium ions than those operating between the bipyridinium ions and citrate stabilized Au NPs surface. Similar behavior is observed with SDS (Figure S2). On the other hand, when presynthesized Au NPs are titrated with EMI, no visible change in the intensity or wavelength of citrate (Figure S3a) or CTAB stabilized (Figure S3b) Au NPs is observed. It demonstrates that though EMI adsorb on the Au 7865


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Figure 7. Flow diagram depicting the selective adsorption of protein fractions on IL coated NPs.

next two bands are due to dimerization or oligomerization.35 The unfolding of zein is related to the amount of SDS used. Usually low amounts of SDS, i.e., in low millimolar concentration range greater than its critical micelle concentration partially reduce the zein.36 Thus, partially reduced protein is prone to the oligomerization due to seeding which is usually more prominent in the case of water insoluble proteins because they contain larger proportions of the nonpolar amino acids. Different amounts of BP coated Au NPs, when treated with zein, picked all fractions with relatively much greater amounts of 21.3 and 25.7 kDa fractions rather than the higher ones because they are present in larger amounts and expected to be more hydrophilic in comparison with 45.7 and 66 kDa fractions. The relative amounts of the zein fractions picked by different samples are quite close to one another and do not show any dependence on the amount of both BP as well as EMI (from 10−40 mM) used in the synthesis against a constant amount of Au salt. Rice Protein. When similar experiments are done with rice protein, the results are quite different from those of the zein protein. Rice protein is another predominantly hydrophobic protein just like that of zein protein. It shows two bands at 15.8 and 31.6 kDa (Figure S4).37 It is high in sulfur containing amino acids like cysteine and methionine. Milled rice grain samples soaked in water at room temperature usually do not provide clear bands, but when they were soaked in acetic acid, different bands appeared at 14, 16, 21, 22, 31, and 34 kDa. Fractions of 14 and 16 kDa are the mixtures of albumin and globulin; 22 kDa belongs to globulin,38,39 while 21 and 31−34 kDa correspond to glutelin.40,41 Our band at 15.8 kDa is mainly due to water insoluble globulin which belongs to a category of globular proteins with usually higher molecular mass than albumins. The second band around 31.6 kDa belongs to glutelin which is also preferentially soluble in acidic, basic, or detergent solutions. BP as well as EMI coated samples when

500−600 nm of wavelength range with positive and negative controls along with the corresponding sample photos shown in Figure 6b. Both IL coated NPs induce significant hemolysis that increases with the increase in the dose amount from 25 to 100 μg/mL. The percentage hemolysis = [(sample absorbance − negative control absorbance)/(positive control absorbance − negative control absorbance) × 100] evaluated from the UV− visible spectra for different samples is compared in the corresponding bar graphs (Figure 6c,d). These graphs show that hemolysis increases with the amount of Au NPs with respect to a constant amount of IL and vice versa. This behavior of IL coated NPs is exploited in protein extraction and characterization in the next section. Protein Extraction. Both BP and EMI coated Au NPs have been further used for the protein extraction28−30 from unpurified proteins solutions (Figure 7). To demonstrate this, we have taken aqueous solutions of zein, rice, and serum proteins. Both zein and rice proteins exist in the form of their homologues in aqueous phase, whereas serum is a complex biological mixture of proteins.31,32 Purified samples of IL coated NPs are treated with aqueous solutions of proteins for 1 h. This allows the adsorption of protein on IL coated NPs mainly driven by the electrostatic interactions of the charged adsorbed layer of ILs with aqueous solubilized protein. Protein loaded NPs are first purified and then digested with SDS Page buffer to remove protein corona (Figure 7). The extracted protein thus obtained is analyzed to determine the molecular mass, and the results are presented in Figure 8 while all bands are listed in Table 1. Most of the bands are not sharp and appear diffused. The polypeptides that possess sequences of hydrophobic side chains as in the case of present protein samples may not denature completely and often produce diffuse bands. Zein Protein. The pure zein protein sample showed four bands of 21.3, 25.7, 45.7, and 66 kDa (Figure 8a). The first two major broad bands indicate the presence of α-zein33,34 while the 7866


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Figure 8. (a) Typical SDS Page analysis scans of some of the samples for zein and (b) serum protein fractions. Details of B1, B4, E1, and E4 IL coated NPs are mentioned in the caption of Figure 6. (c) Comparative bar plots of relative amounts of the most abundant proteins picked by different samples. See details in the text.

Table 1. Representative Protein Fractions Picked by BP and EMI Coated Au NPs from Zein, Rice Protein, and Serum Aqueous Solutionsa zein/kDa pure Bl B4 El E4 a

66.0 66.0 66.0 66.0 66.0

45.7

25.7 25.7 25.7 25.7 25.7

rice protein/kDa 21.3 21.3 21.3 21.3 21.3

31.6 63.1 63.1 63.1 63.1

15.8 55.0 55.0 55.0 55.0

serum/kDa 180

122

80.4

180 180

122 122

80.4 80.4

70.1 70.1 70.1 70.1 70.1

67.5 67.5 67.5 67.5 67.5

63.7 63.7 63.7 63.7 63.7

23.9 23.9 23.9 23.9 23.9

Details of the sample labeling is provided in the caption of Figure 6.

substantial amount of oligomerization42,43 which is identical for all IL coated Au NPs samples. The magnitude of the

treated with rice protein produce two bands at 63.1 and 55.0 kDa, and no band appears at 15.8 and 31.6 kDa indicating a 7867


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shape directing agents to produce tiny Au NPs of around 4−8 nm in their nearly monodisperse state which is usually a difficult process to achieve otherwise. BP coated NPs because of their existence in highly aggregated sheetlike structures are easily transferred to the organic phase by simply employing water insoluble ILs, such as BMI, as phase transfer agents. Both BP and EMI coated NPs show their different abilities to extract different fractions of proteins. Thus, protein extraction ability is IL specific and depends on the functionalization of the IL as BP coated NPs extract proteins of low molar mass whereas EMI coated NPs can extract even bigger proteins as well. To make this procedure even more generalized, further experiments with a variety of IL coated NPs are required because different functional groups have different degrees of extraction ability. This should also to be tested with a variety of protein crude samples.

oligomerization seems to be almost equal for both fractions to produce respective protein−protein aggregates of molar masses 63.1 and 55.0 kDa. It is almost double that from 31.6 to 63.1 kDa, almost quadruple that from 15.8 to 55.0 kDa, or the sum of (2 × 15.8 + 31.6) kDa fractions to give an overall value close to 63.1 kDa. In other words, IL coated Au NPs allow the surface deposition of both fractions (15.8 and 31.6 kDa) in different proportions. The driving force for the oligomerization is attributed to the NPs surface adsorbed protein−protein interactions9 among the individual fractions as mentioned above. Serum. Likewise, IL coated NPs are used for the extraction of blood serum proteins (Figure 8b).44 Serum is a clear yellowish colored fluid which is the part of blood. It does not contain white or red blood cells or a clotting factor. It includes various proteins. Albumins constitute 55%, globulins up to 38%, and fibrinogen comprises of 7% of blood proteins, while the remaining 1% are regulatory proteins. Albumins have relatively lower molar masses of close to 66 kDa. Globulins exist in different sizes. The smallest alpha globulins have molecular masses close to 93 kDa, while the bigger ones are the gamma globulins with molar masses close to 1193 kDa. Fibrinogen possesses about 340 kDa. We observed a clear difference in the picking of the protein fractions from serum between BP coated and EMI coated Au NPs. BP coated NPs pick various proteins up to 70 kDa and of lower molar masses, whereas EMI coated NPs also pick much bigger fractions even up to 180 kDa. Several studies have reported stronger interactions between imidazolium functional groups and water-soluble proteins.45−47 The bigger fractions of serum picked by EMI indicate that EMI possesses greater ability than BP in extracting proteins of higher molar masses. It can be attributed to the smaller size of imidazolium than bipyridinium cations because of cyclohexyloxy functional groups which are expected to induce steric hindrances in the course of bipyridinium cations interactions with bigger protein fractions. Relative Amounts of Extracted Proteins. In order to quantify the relative amount of the protein fractions picked by a particular IL coated NP, relative band intensities have been determined by following the standard densitometric analysis and the results have been depicted in Figure 8c. Qualitatively, both BP as well as EMI coated Au NPs demonstrated greater ability to extract the rice protein in comparison to zein or serum which is most probably due to the overall lower molar mass of the rice protein that provides a better surface adsorption ability in comparison to relatively much higher molar masses of zein and serum fractions. This is further supported by the onset of oligomerization of rice protein which produces oligomerized rice protein bands at 63.1 and 55.0 kDa (Table 1). On the other hand, EMI coated NPs seem to be more efficient in extracting zein and serum in comparison to BP coated NPs because the amounts of relative intensities is much higher than extracted by BP coated NPs. This is further related to the presence of cyclohexyloxy functional groups on BP which in fact cause steric hindrances while extracting greater amounts of protein fractions. The difference between the label “1” and “4” for two different samples of the same IL coated Au NPs refers to 10 and 20 mM of the respective IL used in the reaction, and the samples coated with 20 mM of the respective IL are more efficient in extracting the protein fraction rather than those of 10 mM. Concluding Remarks. The present results demonstrate that both BP as well EMI ILs act as excellent reducing as well as

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b01368.

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UV−visible and TEM images (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (P.K.). *E-mail: [email protected] (M.S.B). ORCID

Mandeep Singh Bakshi: 0000-0003-1251-9590 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

These studies were partially supported by financial assistance from UWGB, NAS, Green Bay, and DST under the nanomission research project [ref no. SR/NM/NS-1057/ 2015(G)], New Delhi. G.K.A. thankfully acknowledges financial support provided by the Research and Development Council (RDC) of Newfoundland and Labrador, NSERC, and the Office of Applied Research at CNA.

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