Preparation of a poly(ionic liquid)-functionalized cellulose aerogel and its application in protein enrichment and separation

Carbohydrate Polymers 218 (2019) 154–162

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Preparation of a poly(ionic liquid)-functionalized cellulose aerogel and its application in protein enrichment and separation

T

Liwei Qiana, , Miaoxiu Yanga, Haonan Chena, Yang Xua, Sufeng Zhanga, Qiusheng Zhoua, Bin Hea, ⁎ Yang Baia, Wenqi Songb, ⁎

a b

National Demonstration Center for Experimental Light Chemistry Engineering Education, Shaanxi University of Science and Technology, Xi’an, 710021, Shaanxi, China Key Laboratory of Organic Polymer Photoelectric Materials, School of Science, Xijing University, Xi’an, 710123, Shaanxi, China

ARTICLE INFO

ABSTRACT

Keywords: Polymeric ionic liquid Cellulose aerogel Adsorption Protein isolation

In this work, a novel poly(ionic liquid) with 1-vinyl-3-aminopropyl imidazolium cations was designed and used to modify cellulose aerogels via Schiff base reaction. The poly(ionic liquid) modified cellulose aerogels (PIL-CA) exhibited a well-interconnected porous structure and a high porosity of 86.2%. A zeta potential study showed the PIL-CA had a strong positive potential of more than 65 mV when its pH was below 6. Furthermore, after 350 min of adsorption experiments, the PIL-CA showed a superior adsorption capacity of 918 ± 8 mg g−1 towards bovine serum albumin (BSA) at pH 6 when its concentration was 1.5 mg m L−1. Finally, the PIL-CA was employed for the selective separation of target protein from a real serum sample, obtaining the BSA with high purity of over 98%.

1. Introduction In recent decades, numerous experts and scholars have been devoted to the development of pharmaceutical-grade protein, medical diagnosis, and research on the pathogenesis of diseases based on proteomics. Among these studies, the isolation and purification of proteins has been of great significance. Currently, conventional methods to separate proteins include precipitation (Burgess, 2009), adsorption (Nakanishi, Sakiyama, & Imamura, 2001) chromatography (Asenjo & Andrews, 2009) and membrane chromatographic techniques (Ghosh, 2002). However, a low adsorption capacity and an insufficient separation efficiency remain major drawbacks for the adsorption and membrane chromatographic techniques. Therefore, the development of new materials possessing a large specific surface area and a favorable porous structure for protein enrichment has aroused wide interest. Aerogels are a class of porous materials with a three-dimensional network structure, low density, high specific surface area, and large porosity. Due to their unique physical structure and properties, aerogels have been widely used in wastewater treatment (Jiang, Dinh, & Hsieh, 2017), gas adsorption (Valdebenito et al., 2018), oil-water separation (Feng, Nguyen, Fan, & Duong, 2015), PM 2.5 capture (Zhang, Sun et al., 2018; Zhang, Yang et al., 2018) and protein enrichment (Anirudhan & Rejeena, 2013). With the advent of the global sustainable development trend, promoting the use of natural renewable resources to prepare

⁎

high-efficiency adsorbents, such as cellulose-based aerogels, has become an important research area (Oliveira, Godinho, & Zattera, 2018). However, since it is difficult for the hydroxyl functional groups in cellulose to form a stable bond with the target adsorbate in the aqueous phase, applications of cellulose-based aerogels in this field are limited. Varieties of functional materials are used to modify cellulose aerogels to enhance the interaction with the adsorbate in the aqueous phase. For example, through self-polymerization of dopamine in a cellulose/LiBr solution, Wei, Huang et al. (2018) and Wei, Wang et al. (2018) prepared a cellulosic aerogel with good adsorption capacity for methylene blue (Wei, Huang et al., 2018). Geng et al. reported a thiol-functionalized nanocellulose aerogel adsorbent fabricated via facile freeze-drying of a nanofibrillated cellulose suspension in the presence of mercaptobased silane sols, and the modified aerogel exhibited outstanding adsorption ability and selectivity towards mercury ions (Geng et al., 2017). Ionic liquids (ILs) are a class of molten salts with melting points at or below 100 °C and generally consist of organic cations and inorganic or organic anions. ILs have many unique physicochemical properties, especially structural tunability, which allow researchers to design and synthesize IL materials with special functions according to their own research needs. Consequently, a large number of functional ILs have been developed for direct protein extraction or to improve protein separation ability by modifying substrates (Quental et al., 2015; Wei,

Corresponding authors. E-mail addresses: [email protected] (L. Qian), [email protected] (W. Song).

https://doi.org/10.1016/j.carbpol.2019.04.081 Received 13 February 2019; Received in revised form 25 April 2019; Accepted 26 April 2019 Available online 29 April 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.

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Huang et al., 2018; Zafarani-Moattar, Shekaari, & Jafari, 2018). Martins et al. synthesized a series of imidazole-type ILs with different alkyl pendant groups and used their aqueous solution for extracting phycobiliproteins from red macroalgae (Martins et al., 2016). A sucrose based ionic liquid colloid was proposed by Sahiner et al., and the prepared microgels showed high amount of protein absorbing capabilities towards Lysozyme and BSA (Sahiner and Sagbas, 2018). More recently, a series of anionic poly(ionic liquid) (PIL) were developed by Dang et al., and the obtained PIL exhibited superior adsorption capacities towards proteins (Dang et al., 2017). In order to prepare a biomass adsorbent with high protein separation ability, in this work, a novel PIL called poly(1-vinyl-3-aminopropyl imidazolium bromide), is synthesized and used for the modification of cellulose aerogels. Compared with common IL modifiers, PIL-based modifiers can provide more adsorption sites for cellulose aerogels (Liu, Liang, Shen, & Bai, 2018), thus theoretically achieving superior protein separation capabilities. The prepared aerogels were characterized using Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and zeta potential tests. In addition, the adsorption performances of modified cellulose aerogels towards proteins were evaluated using adsorption experiments, and their adsorption behaviors were investigated using adsorption isotherms and kinetics. Finally, the selectivity of modified cellulose aerogels was also evaluated by extracting bovine serum albumin from a real sample of bovine calf serum.

sample in buffer solution was characterized using a Malvern G3-ID Zeta potential analyzer at a constant ionic strength (I = 0.6). The concentration of the protein solution was determined using a UV-2550 UV–vis spectrophotometer (Shimadzu Co., Ltd.). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed at a constant current of 0.02 A (Bio-Rad, Singapore). An 1100 system with an automatic injector (0–100 μ L) and UV detector from Agilent Technologies (USA) was used for High Performance Liquid Chromatography (HPLC) experiments. 2.3. Synthesis of ionic liquid (IL) monomers IL monomer was synthesized following a reported procedure with minor modification (Kar, Biswas, Rohini, & Bose, 2015): In a threenecked round-bottom flask, 9.05 m L 1-vinylimidazole (0.1 mol) was dissolved in 100 m L ethanol under a nitrogen atmosphere. Subsequently, 26.27 g BPA (0.12 mol) was added to the above solution and refluxed at 80 °C for 24 h. The mixture solution was precipitated with ethyl acetate to form a yellow viscous solid. After washing with 100 mL ethyl acetate to remove the unreacted substance, the desired IL monomer was obtained via vacuum drying at 50 °C for 12 h. The chemical structure of the IL monomer was confirmed by FT-IR, elemental analysis and 1H NMR, and the results are as follows: FT-IR (KBr, cm−1): 3300, 3210, 3052, 2905, 2891, 1615, 1577, 1465, 1350, 1144, 1023, 966, 856, 734; elemental analysis calcd. (%) for C8H15N3Br2 (233): C 41.20, H 6.44, N 18.03. Found: C 41.12, H 6.46, N 17.93; and 1H NMR spectrum is shown in Fig. S1.

2. Materials and methods 2.1. Materials

2.4. Synthesis of poly(ionic liquid) (PIL)

Cellulose (cotton linter pulp, DP = 600) with α-cellulose content above 95% was provided by Tianjin Zhongchao Paper Co., Ltd. 1-vinylimidazole, 3-bromopropylamine hydrobromide (BPA), epichlorohydrin (ECH), ovalbumin (OVA, pI = 4.5, 43.0 kDa), papain (pI = 8.7, 23.4 kDa) and bovine hemoglobin (Hb, pI = 6.9, 65.0 kDa) were purchased from Sigma-Aldrich Co., Ltd. (China). Bovine serum albumin (BSA, pI = 4.7, 66.2 kDa), lysozyme (Lyz, pI = 11.2, 14.4 kDa) and bovine calf serum (BCS) were purchased from Alfa Aesar Chemical Co., Ltd. (China). Sodium periodate and azobisisobutyronitrile (AIBN) were purchased from Tianjin Kemiou Chemical Reagent Co., Ltd. Sodium cyanoborohydride was purchased from Sinopharm Chemical Reagent Co., Ltd. Acetonitrile (ACN) and trifluoroacetic acid (TFA) were chromatographic grade and purchased from Germany Merck Co., Ltd. All other chemical reagents were of at least analytical grade and were used as received.

PIL was synthesized via free-radical polymerization of the IL monomer. In a typical polymerization, a 100 m L flask was equipped with a magnetic stirrer. Then, 6.26 g IL monomer (0.02 mol) was dissolved in 60 mL DMF with the subsequent addition of AIBN (0.03 g). The mixed solution was deoxygenated by purging with nitrogen for 1 h, and the polymerization was carried out at 70 °C for 12 h (Hu et al., 2017). After the reaction time was complete, the crude product was precipitated as a faint yellow oily liquid by using 100 mL freezing diethyl ether. Subsequently, the crude product was dissolved in 5 mL water and dialyzed by using tubing with a molecular cutoff of 0.5 kDa for 3 days. The aqueous solution containing the product was freezedried, and 5.83 g of the desired PIL was obtained. The chemical structure of PIL was confirmed by 1H NMR and GPC, as shown in Fig. S2. 2.5. Preparation of dialdehyde cellulose aerogel

2.2. Instrumentation

Regenerated cellulose aerogels (R-CA) were fabricated according to previously reported work (Qiu et al., 2018). Briefly, a cellulose solution (3 wt%) was prepared by dissolving cotton linter pulp in a −12.5℃ precooled 7 wt% NaOH/12 wt% urea aqueous solution. Then, 0.3 m L ECH was added to the cellulose solution under vigorous stirring. Finally, the viscous cellulose solution was poured into molds and allowed to stand for 24 h, followed by washing and freeze-drying for 2 days to obtain the R-CA. Dialdehyde cellulose aerogel (D-CA) is obtained from R-CA through a simple oxidation process as follows: 2 g R-CA was added to an 18 m L aqueous solution containing sodium periodate (22 g L−1). The mixture reacted at 50℃ in the dark for 5 h (Li, Chen, Chen, Wan, & Tian, 2018; Li, Kang et al., 2018; Li, Lu et al., 2018; Li, Wu et al., 2018). After the remaining periodate was decomposed by adding an excess of ethylene glycol, the product was washed with ethanol and then freeze-dried for 2 days to obtain the D-CA. In addition, the aldehyde content of D-CA is 11.2 m mol g−1, which was determined following a reported procedure and calculated according to the following equation (Munster et al., 2018):

1

H nuclear magnetic resonance (1H NMR) was applied to investigate the chemical structure of PIL on a Mercury VX-300 spectrometer (Varian) using tetramethylsilane as an internal standard. The molecular weight of PIL was characterized on a Waters 2695 gel permeation chromatography (GPC) system in water with a flow rate of 1 m L min−1. Elemental analysis data were measured by an organic elemental analyzer (Vario MAX, Germany). FT-IR spectra were recorded on a Bruker VERTEX 70 spectrometer. SEM and energy dispersive spectroscopy (EDS) images were recorded using a Tescan VEGA 3 Easy Probe. XPS was performed on a KRATOS AXIS Supra spectrometer for surface analysis with a monochromatized Al-Kα source (hυ = 1486.6 eV) during analysis. X-ray diffraction (XRD) patterns were recorded by Cu Kα radiation over the 2θ range 10-60° using a Bruker D8 Advance instrument. Thermogravimetric (TG) curves were recorded using a Bruker thermogravimetric analyzer in the range 30–800 °C at a constant rate of 10 °C min−1 under a nitrogen atmosphere. The total intrusion volume and porosity were evaluated by mercury intrusion porosimetry (Thermo Fisher) at a pressure range of 0.01–200 MPa. The zeta potential of each 155

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[CHO] =

VNaOH•CNaOH mDACA

2.10. Adsorption models of aerogels

(1)

To investigate the adsorption mechanism of R-CA and PIL-CA towards BSA, the kinetic curves were further analyzed and fitted using pseudo-first-order and pseudo-second-order models, respectively. Their equations are expressed as follows:

where [CHO] is the aldehyde content, mol g−1; ΔVNaOH is the difference of volume of the NaOH solution consumed by the sample titration and the volume of the NaOH solution titrated by the blank sample, L; CNaOH is the concentration of NaOH solution; and mD-CA is the quality of the DCA, g.

ln (Qe

PIL modified cellulose aerogel (PIL-CA) was prepared by Schiff base reaction. The specific method is as follows: 12.5 g PIL was dissolved in 20 m L 0.1 M buffer solution at a pH of 3. After dissolution, 2 g D-CA was added to the above PIL solution under stirring for 7 h at room temperature. Then, sodium cyanoborohydride (1.5 g) was added into the above mixture (Ruan, Stromme, & Lindh, 2018). After a 2 h reaction, the product was washed with 100 mL ethanol and then freeze-dried to obtain the final PIL-CA. The PIL grafting efficiency was 37.4%, which was calculated by the following equation (Xu et al., 2015):

m1

m0 m2

× 100%

(2)

C C 1 = + q qm KL qm ln q = ln KF +

2.7. Acid and alkali tolerance measurements of PIL-CA

2.12. Selective adsorption of aerogels The selective adsorption experiments were measured using a binary mixed protein solution, and Lyz was chosen as a reference. In a typical adsorption experiment, 10 mg PIL-CA was suspended in PBS buffer (10 m L, 0.1 M, pH 6.0) containing different ratio of BSA and Lyz mixture at room temperature. After 350 min, the original and post-adsorption solution were further analyzed by SDS-PAGE. (Guo et al., 2018). The SDS-PAGE assay was conducted using a 12% resolving gel and 5% stacking gel with a standard discontinuous buffer system following the reported procedure. In the real sample test, BCS was diluted 20-fold with PBS buffer (0.1 M, pH 6.0) (Liu et al., 2018). Then, 20 mg PIL-CA was conditioned in the BCS for 12 h. After adsorption was completed, the PIL-CA was treated with 1 M NaCl solution to elute the adsorbed proteins. The elution process was continued until no characteristic peaks of these proteins were observed in the UV region of the spectrum. The eluate was combined, desalted using dialysis tubing with a molecular cutoff of 0.5 kDa, and then lyophilized. The obtained products were dissolved in 5 m L PBS buffer (0.1 M, pH 6.0) and used for running SDS-PAGE electrophoresis with the same method (Qian et al., 2017). In addition, the purity of enriched protein was further characterized with HPLC at 25 °C. The HPLC analysis was performed on a C18 column and the mobile phases were as follow: 0.1% TFA in water (A) and 0.1% TFA in ACN (B), with gradient elution. The analytical flow rate was 1.0 m L min−1 and the wavelength was 214 nm.

(3)

where m is the weight (g) and V is the volume (m3) of the aerogel. 2.9. Adsorption optimization experiments To evaluate the uptake of BSA, 10 mg samples of adsorbents were mixed with 10 mL BSA dissolved in 0.1 M buffer solutions at different pH values. The BSA concentration in the solutions was in the range of 0.1–2 mg m L−1. At specified time intervals, a small portion of a solution was removed and assayed by UV spectroscopy at a wavelength of 280 nm. The amount of BSA adsorbed in the samples was determined from a standard calibration curve and calculated by the following equations (Liu, Huang, Huo, & Gu, 2017; Yi et al., 2017):

Ct ) V m

(8)

The adsorption experiments for the aerogels were also performed towards different proteins, involving OVA, BSA, Hb, Lyz, and papain. All of the above experiments were carried out under optimal adsorption conditions for each protein.

The volumetric mass density of the aerogels can be determined by the volume-mass method and calculated according to the following equation (Li, Chen et al., 2018):

(C0

1 ln C n

(7)

2.11. Adsorption performance of aerogels

2.8. Calculation of the density

Q=

(6)

where qm (mg g−1) is the calculated maximum adsorption capacity, KL (m L mg−1) is the Langmuir constant related to binding affinity, and KF (mg g−1) is the Freundlich constant related to adsorption capacity.

Chemical stabilities involving the acid and alkali tolerance of the PIL-CA were measured according to the following steps: 100 mg PIL-CA samples were placed in 20 m L 0.1 M buffer solutions with pH values ranging from 3 to 11. After stirring for 96 h at room temperature, the samples were thoroughly washed followed by freeze-drying for 2 days and weighed again (Lv et al., 2017).

m V

(5)

where q1 (mg g−1), q2 (mg g−1), qt (mg g−1) and Qe (mg g−1) are the calculated maximum adsorption capacity, adsorption capacity at a certain time and experimental adsorption capacity, respectively; k1 (min−1) and k2 (10−5 g mg−1 min−1) are the rate constants of the pseudo-first-order and pseudo-second-order kinetics models, respectively. To further study the adsorption behavior of R-CA and PIL-CA, the Langmuir model and Freundlich model were employed in this study and expressed as follows:

where m0, m1 and m2 are the weight (g) of D-CA, PIL-CA and PIL, respectively.

=

tk1

t 1 t = + qt q2 k2 q22

2.6. Preparation of PIL modified cellulose aerogel

grafting efficiency(%E ) =

qt ) = ln q1

(4)

where C0 (mg g−1) is the initial concentration of BSA, Ct (mg g−1) is the concentration of BSA after a certain period of adsorption time, V (L) is the volume of BSA solution used, and m (g) is the weight of adsorbent. All experiments were performed in triplicate at room temperature. Besides, the adsorption experimental data are averages and the data error represent standard deviations. 156

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Scheme 1. Schematic illustration of the preparation of PIL-CA and its protein selective adsorption process.

3. Results and discussion

shown in Fig. S3 (a), R-CA exhibits a one-stage weight loss starting at 293 °C corresponding to the loss of the cellulose structure; in contrast, D-CA shows two mass-loss stages: one between 175 °C and 280 °C, which is attributed to the thermal decomposition of the hemiacetal structure formed between aldehyde groups of D-CA, and a second between 280 °C and 350 °C, which is attributed to the cellulose structure (Yan et al., 2018). The lower temperature of 175 °C (T5%) in comparison with R-CA might be attributed to the periodate oxidation modification of cellulose (Xu, Wang, Jin, Wang, & Qin, 2017). After grafting PIL, the PIL-CA exhibited better thermal stability (T5%= 248 °C) than the D-CA because of the higher decomposition temperature (T5%= 420 °C) of PIL. In addition, compared to those of R-CA and D-CA, the TGA curve of PIL-CA revealed a larger amount of residue left after the thermal treatment owing to the immobilization of PIL, which reconfirmed the successful grafting of PIL on the R-CA (Qian, Lei et al., 2018; Qian, Yang et al., 2018). In addition, XRD was employed to detect the crystal structure change of aerogel before and after modification. As shown in Fig. S3 (b), the XRD curve of PIL-CA was almost same as R-CA and D-CA, suggesting that the modification process has little influence on the crystal structure of cellulose aerogel. The morphologies and three-dimensional structure of aerogels were characterized by SEM. It can be clearly seen from Fig. 2(a) that the RCA has a well-connected porous structure, and the pore sizes are approximately 30–70 μm. Compared with R-CA, the surface morphology of D-CA did not change significantly and maintained an interconnected network, as shown in Fig. 2(b). However, modification with PIL had a significant effect on the surface morphology of D-CA. After grafting with PIL, the pore number and size of PIL-CA were obviously decreased (Fig. 2(c)). In addition, high-resolution SEM images and EDS were employed to further characterize the surface morphology and elemental changes of the aerogel (Jiang & Hsieh, 2017). As shown in Fig. 2(d–f), compared to that of R-CA and D-CA, the surface of PIL-CA became rougher, additionally, a new element N appeared and distributed on the surface, further indicating that PIL had been successfully grafted onto the R-CA. To determine the porous structure of the cellulose aerogel, the porosity and total intrusion volume were estimated and are shown in Table 1. Compared to those of R-CA and D-CA, the porosity and total intrusion volume of PIL-CA decreased significantly, which is consistent with the SEM results. However, the PIL-CA still possessed high porosities of more than 86%, which is beneficial to adsorption. Additionally, as shown in the results of the density tests, although the density of PILCA increased relative to that of R-CA and D-CA, it was still in the range expected for cellulose aerogel (Druel, Niemeyer, Milow, & Budtova, 2018; Nissilä, Karhula, Saarakkala, & Oksman, 2018). Since the adsorption capacity is highly dependent on the surface properties of the adsorbent, the zeta potential was investigated at different pH values, and the results are illustrated in Fig. 3. The zeta potential for both R-CA and D-CA exhibited negative values in the pH

3.1. Design of cellulose aerogels In this paper, the design of PIL used to modify cellulose aerogel is based on two considerations. On the one hand, the ionized imidazole functional groups and amino side groups of PIL can generate abundant multiple interactions with proteins such as electrostatic interactions, ππ stacking and hydrogen bonding. On the other hand, the existence of the amino side groups in PIL allows it to be readily grafted onto the surface of the cellulose aerogel by a Schiff base reaction, as shown in Scheme 1. The prepared modified cellulose aerogel theoretically not only has a favorable total intrusion volume and porosity but also contains a large number of adsorption sites, thereby exhibiting excellent protein separation capabilities. 3.2. Characterization of cellulose aerogels The chemical structures of the cellulose aerogel before and after modification were analyzed using FT-IR spectroscopy as shown in Fig. 1(a). The spectrum of R-CA shows typical absorption bands of the cellulose backbone at 1022, 2906 and 3440 cm−1, which are attributed to CeOeC, −CH2−, and −OH bonds, respectively (Zhang, Sun et al., 2018; Zhang, Yang et al., 2018). Compared to that of R-CA, the new peak at 1740 cm−1 in the spectrum of D-CA indicates that carbonyl groups were generated after periodate oxidation, while the disappearance of this peak in the spectrum of PIL-CA illustrates the Schiff base reaction between D-CA and PIL (Li, Chen et al., 2018; Li, Kang et al., 2018; Li, Lu et al., 2018; Li, Wu et al., 2018). Moreover, compared to R-CA and D-CA, the increased intensity of the PIL-CA absorption bands at 3300 cm−1 is possibly attributed to stretching and bending modes of -NH2 (Yuan et al., 2017). Meanwhile, similar absorption bands at approximately 1614, 1360, and 960 cm−1 were shown in the spectra of both PIL and PIL-CA, corresponding to C]N, CeN and ]CeH stretching vibrations of imidazolium rings, respectively (Hu et al., 2017). Thus, these results suggested that the PIL are grafted onto the R-CA. XPS analyses of R-CA, D-CA and PIL-CA provide important information on the evolution of the surface modification occurring upon grafting of the PIL. As shown in Fig. 1(b) and (c), compared to the R-CA, in the XPS high-resolution spectrum of D-CA, the peak area at 287.8 eV revealed a significant increase that was probably attributed to the existence of C]O photoemissions (Li, Chen et al., 2018; Li, Kang et al., 2018; Li, Lu et al., 2018; Li, Wu et al., 2018). Additionally, compared to the survey scan spectra of R-CA and D-CA, as shown in Fig. 1(d), the spectra of PIL-CA showed a new peak at 399.7 eV that was assigned to the nitrogen atoms of the PIL moieties, further suggesting that the PIL was successfully grafted onto the cellulose skeleton. The modified cellulose aerogels were further evaluated by TG. As 157

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Fig. 1. (a) FT-IR spectra of R-CA, D-CA and PIL-CA; XPS high-resolution C 1s spectra of (b) R-CA and (c) D-CA; (d) XPS wide spectra of R-CA, D-CA and PIL-CA.

range from 4 to 10 probably due to the existence of carboxyl groups in the cellulose skeleton. Compared to R-CA and D-CA, PIL-CA exhibits a strong positive charge, and its zeta potential can exceed more than 65 mV when the pH is below 6, which is much higher than that for previously reported cationized cellulose materials (Cai et al., 2018;

Zhang, Dong, Sun, Wu, & Li, 2017). In addition, the tolerance of PIL-CA was investigated in acidic and alkaline environments, as shown in Fig. 3; the results showed that PIL-CA had a high stability at pH values greater than 5, with a mass loss of less than 1%.

Fig. 2. SEM images of R-CA (a), D-CA (b), and PIL-CA (c); High-resolution SEM and EDS images of R-CA (d), D-CA (e), and PIL-CA (f). 158

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reaching equilibrium after 270 min, which suggested stronger interactions between PIL-CA and BSA (Qian, Lei et al., 2018; Qian, Yang et al., 2018). Therefore, in the following adsorption studies, a contact time of 350 min was chosen for both R-CA and PIL-CA. Isothermal experiments were carried out to investigate the effect of BSA concentration on the adsorption capacity of the aerogel. As shown in Fig. 4(c), the adsorption capacity of aerogels increased as BSA concentration increased, until the BSA concentration was 1.5 mg m L−1, and both R-CA and PIL-CA reached saturation adsorption with maximum adsorption capacities of 286 ± 5 mg g−1 and 918 ± 8 mg g−1, respectively, demonstrating the beneficial effect of PIL modification on the cellulose aerogel. Therefore, the optimal adsorption concentration of BSA was selected as 1.5 mg m L−1 for R-CA and PIL-CA. Table S1 lists the previously reported adsorption capacities of adsorbents towards BSA. It can be seen that PIL-CA provided a much higher adsorption ability towards BSA with respect to other materials.

Table 1 Porosity, total intrusion volume and density of the cellulose aerogel. Sample

Porosity (%)

Total Intrusion Volume (mL g−1)

Density (mg cm−3)

R-CA D-CA PIL-CA

90.8 ± 0.6 88.6 ± 0.9 86.2 ± 0.4

6.99 ± 0.09 5.36 ± 0.05 4.54 ± 0.06

15.8 ± 0.3 13.1 ± 0.1 57.7 ± 0.8

3.4. Adsorption mechanism of cellulose aerogels In order to simulate the adsorption process towards BSA, kinetics investigations were conducted through the evaluation of the pseudofirst-order and pseudo-second-order kinetic models. According to previous studies, the pseudo-first-order model is commonly used to describe a physical adsorption process in which there is a linear relationship between the reaction rate and the concentration of a reactant (Xiao et al., 2018), whereas the pseudo-second-order model universally describes chemical sorption between an adsorbent and adsorbate (Gao et al., 2018). As shown in Fig. S4 and Table 2, the pseudo-second-order model fitted the experimental data of PIL-CA better, with a higher correlation coefficient (R2) value than the pseudo-first-order model, and its calculated q2 value was closer to the experimental value, suggesting that the process may be mainly controlled by the chemical adsorption mechanism. In contrast, the q1 value of the pseudo-first-order model for RCA was closer to the experimental data, indicating that the adsorption might be attributed to a physical process (Li, Chen et al., 2018; Li, Kang et al., 2018; Li, Lu et al., 2018; Li, Wu et al., 2018). In addition, Langmuir and Freundlich isotherm model was employed to investigate the adsorption behavior of PIL-CA. Usually, the Langmuir isotherm describes homogeneous adsorption, in which all of the sites have equal affinity for the adsorbate (Hippauf et al., 2016). In contrast, the Freundlich model is an empirical equation based on adsorption on a heterogeneous surface (Zheng, Yang, Meng, & Peng, 2019). As shown in Fig. S5 and Table 3, the R2 of R-CA from the plot of the Freundlich model was nearly 1, indicating that BSA adsorption on R-CA is a heterogeneous adsorption process (Lan, Shao, Wang, & Gu, 2015). Compared to that for R-CA, the adsorption process of BSA on PIL-CA was more consistent with the Langmuir model (R2 ≥ 0.99), indicating that the uptake of BSA occurred on a homogenous surface with monolayer adsorption.

Fig. 3. The zeta potential of R-CA, D-CA and PIL-CA at different pH and the stability of PIL-CA in acidic and alkaline environments.

3.3. Optimization of adsorption conditions of cellulose aerogels The initial pH value of the solution is an important factor in the adsorption process because it affects the ionization behavior and surface charges of the BSA and adsorbent. As shown in Fig. 4(a), R-CA showed a moderate adsorption performance towards BSA when the pH was lower than 5. However, with increasing pH, the adsorption capacity of R-CA towards BSA dropped from 286 mg g−1 to 40 mg g−1, most likely due to electrostatic repulsions (Ji et al., 2018). After modification with PIL, PIL-CA exhibited a superior high adsorption capacity towards BSA at each pH value. In particular, the adsorption capacity of PIL-CA could reach more than 800 mg g−1 when the pH was in the range of 5–7. This may be due to the fact that this pH range is higher than the isoelectric point of BSA, resulting in a strong electrostatic attraction between the protein and the PIL-CA. Further increase of pH, the adsorption capacity decreased most likely owing to the competitive adsorption from hydroxide ion (Zhang et al., 2013). Consequently, considering the pH effect on the tolerance and adsorption capacity, the optimum pH value for PIL-CA was selected as 6, while that for R-CA was selected as 5. To determine the effect of contact time on the adsorption capacity, the adsorption kinetics were studied for both R-CA and PIL-CA. As shown in Fig. 4(b), the adsorption capacity of R-CA and PIL-CA showed the same increasing trend as the contact time increased. However, the binding of BSA on R-CA tended to achieve adsorption equilibrium after 350 min, whereas that on PIL-CA has a faster adsorption profile,

Fig. 4. Effect of (a) initial pH; (b) contact time and (c) initial concentration of BSA solution on the adsorption capacity of R-CA and PIL-CA towards BSA. 159

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Table 2 Parameters of adsorption kinetics of R-CA and PIL-CA. Qe (mg g−1)

Adsorbents

Pseudo-first-order −1

q1 (mg g R-CA PIL-CA

286 ± 5 918 ± 8

)

Pseudo-second-order k1 (min

287 992

−1

)

2

q2 (mg g−1)

k2 (10−5 g mg-1 min-1)

0.999 0.952

327 926

7.38 25.03

R1

0.012 0.013

R22 0.996 0.999

Table 3 Parameters of adsorption isotherms of R-CA and PIL-CA. Adsorbents

Qe (mg g−1)

Langmuir

Freundlich

−1

qm (mg g R-CA PIL-CA

286 ± 5 918 ± 8

415 921

)

−1

KL (mL mg

)

4.43 13.24

RL2

KF (mg g−1) 0.973 0.998

305 1212

1/n

RF2

0.43 0.41

0.999 0.975

Fig. 5. (a) The adsorption capacity of R-CA and PIL-CA towards various kind of proteins; (b) SDS-PAGE analysis of PIL-CA adsorption on mixture solution with different BSA/Lyz ratio: lane 1 and lane 2 are the pure BSA and Lyz solutions at each concentration of 1 mg mL−1, respectively; lane 3 and lane 4 are the mixture solution containing 0.5 mg m L−1 of BSA and 1.5 mg m L−1 of Lyz before and after adsorption using PIL-CA, respectively; lane 5 and lane 6 are the mixture solution containing 1.0 mg m L−1 of BSA and 1.0 mg m L−1 of Lyz before and after adsorption using PIL-CA, respectively; lane 7 and lane 8 are the mixture solution containing 1.5 mg m L−1 of BSA and 0.5 mg m L−1 of Lyz before and after adsorption using PIL-CA, respectively; (c) SDS-PAGE analysis of PIL-CA adsorption on real serum sample: lane 1 is a protein molecular weight marker; lane 2 and lane 3 are BCS before and after adsorption using PIL-CA, respectively; and lane 4 is the protein eluted from PIL-CA; (d) Chromatographic analysis of the enriched proteins. 160

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3.5. Adsorption performance of cellulose aerogels

for Experimental Light Chemistry Engineering Education (Shaanxi University of Science and Technology) (Grant no. 2018QGSJ02-18).

To further investigate the adsorption performance of cellulose aerogel, adsorption experiments were carried out on various proteins with different pI and Mw values. As shown in Fig. 5(a), R-CA showed moderate and nonspecific adsorption capacity towards all five kinds of proteins due to its porous structure and hydrogen bonding interactions with proteins. Compared to R-CA, PIL-CA exhibited various adsorption performances for different proteins. In particular, PIL-CA had a better adsorption performance towards negatively charged proteins, whereas its adsorption capacity towards Hb, Lyz and papain were relatively low. The above experiments showed that grafting PIL was beneficial for enhancing the selective separation ability of cellulose aerogel towards protein.

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2019.04.081. References Anirudhan, T. S., & Rejeena, S. R. (2013). Selective adsorption of hemoglobin using polymer-grafted-magnetite nanocellulose composite. Carbohydrate Polymers, 93(2), 518–527. Asenjo, J. A., & Andrews, B. A. (2009). Protein purification using chromatography: Selection of type, modelling and optimization of operating conditions. Journal of Molecular Recognition, 22(2), 65–76. Burgess, R. R. (2009). Protein precipitation techniques. Methods in Enzymology, 463, 331–342. Cai, Y., Su, S., Navik, R., Wen, S., Peng, X., Pervez, M. N., et al. (2018). Cationic modification of ramie fibers in liquid ammonia. Cellulose, 25(8), 4463–4475. Dang, M., Deng, Q., Fang, G., Zhang, D., Liu, J., & Wang, S. (2017). Preparation of novel anionic polymeric ionic liquid materials and their potential application to protein adsorption. Journal of Materials Chemistry B, 5(31), 6339–6347. Druel, L., Niemeyer, P., Milow, B., & Budtova, T. (2018). Rheology of cellulose-[DBNH] [CO2Et] solutions and shaping into aerogel beads. Green Chemistry, 20(17), 3993–4002. Feng, J., Nguyen, S. T., Fan, Z., & Duong, H. M. (2015). Advanced fabrication and oil absorption properties of super-hydrophobic recycled cellulose aerogels. Chemical Engineering Journal, 270, 168–175. Gao, K., Su, Y., Zhou, L., He, M., Zhang, R., Liu, Y., et al. (2018). Creation of activepassive integrated mechanisms on membrane surfaces for superior antifouling and antibacterial properties. Journal of Membrane Science, 548, 621–631. Geng, B., Wang, H., Wu, S., Ru, J., Tong, C., Chen, Y., et al. (2017). Surface-tailored nanocellulose aerogels with thiol-functional moieties for highly efficient and selective removal of Hg(II) ions from Water. ACS Sustainable Chemistry & Engineering, 5(12), 11715–11726. Ghosh, R. (2002). Protein separation using membrane chromatography: Opportunities and challenges. Journal of Chromatography A, 952(1-2), 13–27. Guo, Z. Y., Hai, X., Wang, Y. T., Shu, Y., Chen, X. W., & Wang, J. H. (2018). Core-corona magnetic nanospheres functionalized with zwitterionic polymer ionic liquid for highly selective isolation of glycoprotein. Biomacromolecules, 19(1), 53–61. Hippauf, F., Huettner, C., Lunow, D., Borchardt, L., Henle, T., & Kaskel, S. (2016). Towards a continuous adsorption process for the enrichment of ACE-inhibiting peptides from food protein hydrolysates. Carbon, 107, 116–123. Hu, F., Fang, C., Wang, Z., Liu, C., Zhu, B., & Zhu, L. (2017). Poly (N-vinyl imidazole) gel composite porous membranes for rapid separation of dyes through permeating adsorption. Separation and Purification Technology, 188, 1–10. Ji, Y. L., An, Q. F., Weng, X. D., Hung, W. S., Lee, K. R., & Gao, C. J. (2018). Microstructure and performance of zwitterionic polymeric nanoparticle/polyamide thin-film nanocomposite membranes for salts/organics separation. Journal of Membrane Science, 548, 559–571. Jiang, F., & Hsieh, Y. L. (2017). Cellulose nanofibril aerogels: Synergistic improvement of hydrophobicity, strength, and thermal stability via cross-linking with diisocyanate. ACS Applied Materials & Interfaces, 9(3), 2825–2834. Jiang, F., Dinh, D. M., & Hsieh, Y. L. (2017). Adsorption and desorption of cationic malachite green dye on cellulose nanofibril aerogels. Carbohydrate Polymers, 173, 286–294. Kar, G. P., Biswas, S., Rohini, R., & Bose, S. (2015). Tailoring the dispersion of multiwall carbon nanotubes in co-continuous PVDF/ABS blends to design materials with enhanced electromagnetic interference shielding. Journal of Materials Chemistry A, 3(15), 7974–7985. Lan, T., Shao, Z.q., Wang, J.q., & Gu, M.j. (2015). Fabrication of hydroxyapatite nanoparticles decorated cellulose triacetate nanofibers for protein adsorption by coaxial electrospinning. Chemical Engineering Journal, 260, 818–825. Li, N., Chen, W., Chen, G., Wan, X., & Tian, J. (2018). Low-cost, sustainable, and environmentally sound cellulose absorbent with high efficiency for collecting methane bubbles from seawater. ACS Sustainable Chemistry & Engineering, 6(5), 6370–6377. Li, J., Kang, L., Wang, B., Chen, K., Tian, X., Ge, Z., et al. (2018). Controlled release and long-term antibacterial activity of dialdehyde nanofibrillated cellulose/silver nanoparticle composites. ACS Sustainable Chemistry & Engineering, 7(1), 1146–1158. Li, F., Lu, L., Gao, D., Wang, M., Wang, D., & Xia, Z. (2018). Rapid synthesis of threedimensional sulfur-doped porous graphene via solid-state microwave irradiation for protein removal in plasma sample pretreatment. Talanta, 185, 528–536. Li, Z., Wu, H., Yang, M., Xu, D., Chen, J., Feng, H., et al. (2018). Stability mechanism of O/W pickering emulsions stabilized with regenerated cellulose. Carbohydrate Polymers, 181, 224–233. Liu, J., Liang, Y., Shen, J., & Bai, Q. (2018). Polymeric ionic liquid-assembled grapheneimmobilized silica composite for selective isolation of human serum albumin from human whole blood. Analytical and Bioanalytical Chemistry, 410(2), 573–584. Liu, Y., Huang, H., Huo, P., & Gu, J. (2017). Exploration of zwitterionic cellulose acetate antifouling ultrafiltration membrane for bovine serum albumin (BSA) separation.

3.6. Selective adsorption experiments To further demonstrate the selective adsorption capacity of PIL-CA, the mixture solution of BSA/Lyz and a real sample of BCS were chosen respectively in selective binding experiments using SDS-PAGE (Lu, Lin, Zhang, & Yao, 2017). As shown in lane 4, 6 and 8 of Fig. 5(b), the band representing BSA approximately disappeared after adsorption by PILCA in comparison of lane 3, 5 and 7, demonstrating that PIL-CA had a good selective adsorption performance towards BSA in different ratio of Lyz/BSA mixed solution. Additionally, the isolation and purification capabilities of PIL-CA towards protein were investigated with adsorption experiments in a real sample of BCS. As shown in Fig. 5(c), the multiple bands in lane 2 revealed the presence of other contaminant proteins in BCS besides BSA. After adsorption by PIL-CA, the color of the band at 66.2 kDa representing BSA in lane 3 was much lighter than that in lane 2, whereas other bands did not change much. Moreover, there was a clear band at 66.2 kDa in lane 4, and the HPLC analysis also showed the purity of enriched protein was more than 98%, as shown in Fig. 5(d), proving the great application potential of PIL-CA in the field of protein separation and enrichment. 3.7. Reusability of PIL-CA Reusability is an important criterion for a material to be applicable in practical purposes. To demonstrate the reusability of PIL-CA, the adsorption-desorption cycle was repeated six times. As shown in Fig. S6, PIL-CA lost only 8.67% of its maximum adsorption capacity even after six cycles, and its mechanical structure was barely changed in SEM images, indicating that PIL-CA exhibited good reusability as an adsorbent. 4. Conclusion In summary, a novel PIL functionalized cellulose aerogel was designed and prepared via a convenient Schiff base reaction. Taking advantage of the properties of the PIL and cellulose aerogel, the as-prepared PIL-CA exhibited a well-interconnected porous structure and a strong positive charge. More importantly, PIL-CA was demonstrated to provide superior adsorption capacity and high selectivity towards BSA. The successful selective separation of BSA from BCS with PIL-CA suggested its great potential and practicability in the field of proteomic analysis, medical diagnosis, and even sensors. Acknowledgments This project was supported by the National Nature Science Foundation of China (Grant no. 21805177), the Special Scientific Research Project of Shaanxi Education Department (Grant no. 18JK0095) and the Open Project of the National Demonstration Center 161

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