Preparation of ionic liquid polymer materials and their recognition properties for proteins

Abstract

In this paper, two polymer materials were synthesized by using 1-allyl-3-butylimidazolium chloride ([ABIM][Cl]) or 1-vinyl-3-octylimidazolium bromide ([VOIM][Br]) as functional monomers, and arcylamide (AAm) as a co-functional monomer. Bovine serum albumin (BSA), bovine hemoglobin (BHb), lysozyme (Lyz), and cytochrome C (Cyt C) that have different properties were selected as model proteins to investigate the absorption and separation abilities of ionic liquids polymer materials. The selective absorption properties of two polymer materials for proteins were compared for the first time. The results showed that [ABIM][Cl] ionic liquid polymer material had a high binding capacity for BHb (828.5 mg g⁻¹), and [VOIM][Br] polymer material possessed a high binding capacity for BSA (804.7 mg g⁻¹). Different proteins can be separated by altering the cations of the ionic liquid. In addition, the structure, morphology and thermal properties of ILs polymer materials were characterized by scanning electron microscope (SEM), fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). These results indicated that the IL polymer materials created a tendency for the partition of proteins efficiently and may be applied in medical diagnosis, proteomics, biotechnology and sensors broadly.

---

Introduction

Protein is a polymer molecule with biological activity in vivo, and plays crucial roles in biological functions like metabolism, gene expression and signal transduction.¹ In addition, many proteins are used in therapeutic and medical diagnosis. Protein separation is one of the important steps for its practical application.¹ In recent years, many methods have been developed for protein separation, including precipitation, chromatography, centrifugation and electrophoresis.² These classical methods are not only costly and time-consuming, but turning into denatured with organic solvent, pH, ionic strength. As a result, some research groups have been devoted to developing new materials for protein separation in recent years. Welsch et al.³ reported the separation of bovine serum albumin (BSA) and lysozyme (Lyz) on two types of well-defined colloidal particles, which were spherical polyelectrolytes and core–shell microgels with a shell of crosslinked poly (N-isopropylacrylamide) (pNiPAm) chains. Zhu et al.⁴ devoted to synthesizing mesoporous organosilicas (PMO) with anorganobridged (–CH₂–) for the absorption of BSA. Porous dextran microspheres with good morphology were synthesized by reversed suspension polymerization for the absorption of BSA.⁵ However, these materials may meet challenges in practical applications for absorbents such as toxic monomers or activators, high back pressure and poor biocompatibility. Consequently, it is highly desirable to explore new absorption materials for separation of proteins with high biocompatibility.

In recent years, ionic liquids (ILs), especially imidazolium-based ILs, have been playing an increasingly important role in chemical synthesis, biocatalytic transformation, electrochemical device designs, analytical and separation process.^6–9 ILs have many attractive properties such as appreciable liquid ranges, low volatilities, thermal stability, selective miscibility, non-flammability and reusability.¹⁰ The physico-chemical properties of ILs may be changed by altering the cationic moieties, which makes it possible for the applications of ILs in various areas.¹¹ Ionic liquids have been used for the separation and absorption of proteins and deoxyribonucleic acid (DNA) with high extraction efficiency.^12,13 Imidazolium-based ILs were utilized to extract BSA, cytochrome C (Cyt C) and γ-globulins.¹⁴ The ionic liquid-based aqueous two-phase extraction of selected proteins was researched by Pei et al.¹⁴ However, there are some disadvantages in liquid–liquid extraction such as time and labor intensive, and the use of large amount of organic solvents. In contrast, solid-phase extraction is a much less reagent-consuming, convenient, safe and efficient approach. Additionally, ILs have also been applied in modified materials for solid-phase extraction and separation of drugs.¹⁵ Therefore, the use of ILs in the modified materials for the separation of proteins is possible in the future.

In this paper, ILs polymer materials were synthesized by using 1-allyl-3-butylimidazolium chloride ([ABIM][Cl]) or 1-vinyl-3-octylimidazolium bromide ([VOIM][Br]) and arcylamide (AAm) as co-functional monomers, and N,N′-methylene bisacrylamide (MBA) as a cross-linker. Scanning electron microscope (SEM), fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimeter (DSC), and thermogravimetric analysis (TGA) were used to characterize different ILs polymer materials. Absorption property of the ILs polymer materials was evaluated by using four typical proteins like bovine hemoglobin (BHb), molecular weight (MW) 64.5 kDa, isoelectric point (6.8), BSA (MW 67.0 kDa, pI 4.8), Lyz (MW 14.4 kDa, pI 11.2) and Cyt C (MW 12.4 kDa, pI 9.8) as model proteins.

Experimental

Materials

The following model proteins, BHb, BSA, Lyz, Cyt C were purchased from Sangon Biotech Co., Ltd. (Shanghai, China), and used without further purification. AAm (≥98.0 wt%) and MBA were purchased from Tianjin Chemical Reagent Co., Ltd. (Tianjin, China). [VOIM][Br] was purchased from Beijing Hwrk Chem Co., Ltd. (Beijing, China). N,N,N′N′-tetramethylethylenediamine and sodium dodecyl sulfate (SDS) used in this study were purchased from Bio Basic Inc (Shanghai, China). 1-Allyl imidazole was acquired from Sigma-Aldrich Inc (Shanghai, China). 1-Chlorobutane (GR grade), trifluoroacetic acid and HPLC-grade acetonitrile were purchased from Kangkede Technology Co., Ltd (Tianjin, China). Ammonium persulfate (APS) and acetic acid glacial were analytical grade and purchased from Tianjin Chemical Reagent Co., Ltd. (Tianjin, China). All other chemicals were of analytical grade and commercially available, including acetic acid glacial, boric acid, citric acid and oxalic acid dehydrate.

Synthesis of 1-allyl-3-butylimidazolium chloride

The [ABIM][Cl] was prepared according to the procedure described in the literature.¹⁶ 1-Chlorobutate (7.65 g, 0.1 mol) was added to 9.55 g (0.077 mol) of 1-allylimidazole. The solution was refluxed for 3 h at 50 °C under stirring. The excess 1-allyaimidazole was removed by rotary evaporation. A viscous light yellow product was obtained, which was vacuum-heated to remove the remaining water.

Synthesis of IL polymer materials

The polymer materials were prepared by the procedures described in the literature.¹⁷ AAm (0.57 g), MBA (0.030 g) and a certain amount of ILs ([ABIM][Cl] or [VOIM][Br]) were dissolved in 7.2 mL of sodium phosphate buffer solution (PBS, 10 mM, pH 6.8), and reacted for 1 h at 25 °C under stirring. The oxygen was removed by nitrogen bubbling for 5 min. 200 μL of 10% APS (w/v) and 100 μL of 5% TEMED (v/v) were then added to the mixed solution. The polymerization reaction was carried out at 25 °C for 24 h. After the reaction, the resultant ILs polymer materials were cut into small disks and the remaining reactants were washed with deionized water. The ILs polymer materials were freeze-dried in a Virtis freeze drier to remove the remaining water and then ground into particles through an 80 mesh sieve for further study.

Protein adsorption

The adsorption property of polymer materials for BHb was studied under different pH values (6.0–8.0) with the concentration of BHb at 1.0 mg mL⁻¹. 5.0 mg of ILs polymer material was added to 5.0 mL protein buffer solution, and shaken at room temperature for 12 h to ensure absorption equilibrium. The absorption amount was calculated by the following formula:
where Q is the mass of protein absorbed per gram of ILs materials, C_i and C_f (mg mL⁻¹) are the initial concentration protein and the final concentration protein, respectively. V (mL) is the volume of protein solution and W (g) is the weight of the ILs polymer material.

In adsorption dynamics experiment, 5.0 mg of ILs polymer materials were mixed with 5.0 mL of protein buffer solution (PBS, 10 mM, pH 6.8) with an initial BHb concentration of 1.0 mg mL⁻¹, and then shaken at room temperature. At different time intervals, the amount of BHb adsorbed by ILs polymer materials was measured by UV-VIS spectrophotometer.

In the experiment equilibrium binding isotherms, the BHb concentration ranged from 0.6 to 1.4 mg mL⁻¹. 5.0 mg of ILs polymer materials were mixed with BHb solution, and shaken for 12 h at room temperature. The concentration of BHb in the solution was measured by ultraviolet visible (UV-VIS) spectrophotometer.

In the experiment of selective absorption, BHb, BSA, Lyz, and Cyt C were chosen as model proteins. The batch absorption tests including BHb, BSA, Cyt C, Lyz, and the binary mixtures of Lyz–BSA, and the ternary mixtures of Lyz–BSA–Cyt C, and the quaternary mixtures of Lyz–BSA–Cyt C–BHb were investigated in the experiment. In each adsorption experiment, 5.0 mg of ILs polymer materials were added to 5.0 mL of buffer solution (PBS, 10 mM, pH 6.8) with the concentration of BHb at 1.0 mg mL⁻¹. The mixture was shaken for 12 h at room temperature. The concentration of protein was measured by high performance liquid chromatography (HPLC).

The experiments were performed by a Shimadzu 1210 series HPLC unit with a Hypersil 300A-C₈ 4.6 × 150 mm, 5 μm reversed-phase column. The protein was detected at a wavelength of 220 nm with a ultraviolet (UV) detector. Two mobile phases were used: (A) ultrapure water with 0.1% trifluoroacetic acid (TFA) and (B) acetonitrile with 0.1% TFA for the linear gradient elution. The chromatograms were obtained under gradient elution at a flow rate of 0.8 mL min⁻¹ at 35 °C. The eluting condition of solvent B increased from 20 to 70% (v/v) in 20 min.

Protein desorption

The polymers adsorbed proteins was washed with 1 mL PBS (10.0 mM, pH = 6.8) to remove the free proteins on the surface of the polymers. Then, 2 mL PBS (10 mM, pH = 6.8) containing NaCl (0.1 mol) and 2 mL acetonitrile/water (7/3, v/v) containing TFA (0.1%) were added and shaken at room temperature for 24 h. After centrifuging, the concentration of protein in supernatant was measured by HPLC to determine the desorption ratio.

Results and discussion

Synthesis of ILs polymer materials

ILs could be used widely as extractant for the extraction and purification of catalytically active enzymes and model proteins. Importantly, the catalytic activity and conformation of proteins were not changed after extraction in the IL-based aqueous two-phase systems^18–22 in the biochemical field. Poly-acrylamide (PAAm) that resembled natural living tissue could imbibe large amounts of water by three-dimensional, hydrophilic, polymeric network structures.^23,24 Consequently, in our research, [ABIM][Cl] or [VOIM][Br] and AAm as co-functional monomers, MBA was chosen as crosslinker for the synthesis of ILs polymer materials. As IL was composed of anions and cations, anions in the eluents might affect the adsorption capacity of the polymer materials. Hence, in this study, the ILs polymer materials were washed with different eluents and then with deionized water to remove the remaining eluents. As can be seen from Fig. 1, the [ABIM][Cl] polymer material that was washed with a 10% (v/v) acetic acid (AcOH) solution containing 10% (w/v) SDS (AcOH–SDS) showed the highest binding capacity of about 828.5 mg g⁻¹ for BHb, while the [VOIM][Br] polymer material washed with deionized water showed the highest binding capacity of 615.2 mg g⁻¹ for BHb.

Fig. 1 Effect of anions on the adsorption of the ILs polymer materials toward BHb. Adsorption conditions: time = 12 h, m = 5.0 mg, V = 5.0 mL, C₀ = 1.0 mg mL⁻¹, (PBS, 10 mM, pH = 6.8). All values are means of three measurements.

The absorption capacity for BHb reduced after eluting with acids. The reduced acidity resulted in the decreased ability of acid to provide proton, which was of great benefit for the formation of hydrogen bonds. The absorption capacity for protein after eluting with NaCl was lower than with AcOH–SDS solution and deionized water. We speculated that the electrostatic repulsive-force was increased with increasing ion strength. The absorption capacity for protein after eluting with AcOH–SDS solution was higher than with other eluents. Eluting with AcOH–SDS solution was of great benefit to the formation of hydrogen bonds. Yuan et al.¹⁷ found that [VBIM][Cl] and AAm polymer materials washed with 10% (v/v) AcOH solution containing 10% (w/v) SDS showed much larger adsorption capacity for protein than unwashed. The high adsorption capacity of the chitosan/PAA polymer materials for protein was also observed by Fu et al.²⁵ They speculated that the inter-and intra-molecular hydrogen bonds were the main driving forces. However, [VOIM][Br] polymer materials washed with AcOH–SDS solution had the maximum absorption capacity of 391.6 mg g⁻¹ for BHb. It was less than that of the one washed with deionized water.

The extraction efficiency and the hydrophobicity of the ILs were reported to increase with increasing alkyl chain length of cations of the imidazolium ILs.¹ In this study, the adsorption capacity of [ABIM][Cl] polymer material for BHb was much larger than that of [VOIM][Br] polymer material. An increase of polymer chain length usually decreases the partition coefficient due to size-exclusion effects.²⁶ Our results indicated that the ILs polymer materials absorbed proteins by multiple interactions rather than only by hydrophobic interaction.

Characterization

The morphology of the ILs polymer materials was characterized by SEM (Fig. 2). M₁ was synthesized at the molar ratio of [ABIM][Cl]–AAm of 1 : 10 and the eluent was AcOH–SDS solution. The synthesized condition of M₁ was that the molar ratio of [ABIM][Cl]–AAm = 1 : 10 and the eluent was AcOH–SDS solution, M₂ was synthesized with the molar ratio of [ABIM][Cl]–AAm = 1 : 10 and washed with deionized water, M₃ was synthesized with the molar ratio of [VOIM][Br]–AAm = 1 : 20 and washed with AcOH–SDS solution, and M₄ was synthesized with the molar ratio of [VOIM][Br]–AAm = 1 : 20 and washed with deionized water. As can be seen from Fig. 2, all the polymer materials were porous but different in morphology. There were a large number of well-distributed and continuous interconnected pores in M₁ and M₂. The pore size of M₁ was much larger than the size of protein molecules, hence leading to the easy pass of proteins through the pores.²⁷ M₁ showed macropores and compact homogeneous network structure (Fig. 2(a)). M₂ showed a heterogeneous morphology with pores comprised of various sizes. The structure and morphology of M₃ were heterogeneous, while M₄ has compact structure. However, there were not nanopores from the morphologic images of the materials. The main reason may be attributed to the materials shrink caused by freeze-dried.

Fig. 2 SEM images of different materials (×3000). (a) M₁, (b) M₂, (c) M₃, (d) M₄.

The presence of functional groups or chemical bonds were identified by FT-IR spectra in Fig. 3(a) and (b). From Fig. 3(a), the band at 3187.30 cm⁻¹ for =C–H stretching in imidazolium ring of [VOIM][Br] was observed. [VOIM][Br] exhibited bands at 2928.79 cm⁻¹ and 1683.50 cm⁻¹ for stretching vibration of C–H and C=N in imidazolium ring, respectively, and band at 633.27 cm⁻¹ for =C–H bending vibration. From Fig. 3(b), the band at 3418.31 cm⁻¹ for the stretching vibration of NH₂ group in PAAm was detected. Besides, the band attributed to the alkyl-butyl-imidazolium cation was observed at 2930.53 cm⁻¹. Furthermore, in order to investigate the chemical structure of the polymer, the resulted polymers were characterized by elemental analysis and X-ray photoelectron spectroscopy (XPS), respectively. The results of the elemental analysis were showed in Table 1. The results of XPS showed that the Cl% (atm.%) was determined as 1.44%.

Fig. 3 FT-IR spectra of (a) [VOIM][Br] and (b) [ABIM][Cl].

Table 1 The elemental content (N, C, H and O) of the polymer

Polymers	N (%)	C (%)	H (%)	O (%)
Polymer contained ILs	16.54	46.05	7.31	26.40
Polymer uncontained ILs	17.28	45.82	7.04	29.82

---

The result of DSC is shown in Fig. 4. With the temperature increasing from 30 to 350 °C, all the materials presented two endothermic peaks. The peak near 100 °C was due to the pneumatolysis of moisture in the materials. The melting temperature (T_m) of M₁, M₂, M₃ and M₄ were 258.84, 263.54, 260.44 and 260.35 °C, respectively. The result showed that the T_m of M₁ was much lower than that of M₂, indicating that adding [ABIM][Cl] and washed by AcOH–SDS could both diminish the T_m. This could be attributed to the destructive effect of [ABIM][Cl] and the AcOH–SDS solution on the inter- and intra-molecular hydrogen bonds and ionic bonds. However, the T_m of M₃ and M₄ were not different significantly. We speculated that the AcOH–SDS solution had little influence on the absorption capacity of protein onto [VOIM][Br] polymer material.

Fig. 4 DSC thermograms of different materials.(a) M₁, (b) M₂, (c) M₃, (d) M₄.

Besides, we discussed the influence of anions and different ionic liquids on the thermal stability of the ILs polymer materials. The temperature was increased from 25 to 300 °C slowly in TGA. The weight loss curves of M₁, M₂, M₃ and M₄ are exhibited in Fig. 5. The TGA curves can be divided into three stages. The first stage occurring at 40–150 °C was mainly due to the loss of water, which includes bound water and crystal water. No significant differences were observed in the weight loss of ILs polymer materials at this stage. The second stage of weight loss, which started at about 250 °C, could be attributed to the degradation of polymer chains. The weight loss of M₁ was lower than other materials. The third stage all occurred at about 380 °C, with the weight loss being as 73.3%, 80.9%, 76.5% and 77.1% for M₁, M₂, M₃ and M₄, respectively. The results showed that when the materials were washed with AcOH–SDS, the presence of different ionic liquids had little influence on the weight losses in this stage. However, the weight losses of materials were obvious at 500 °C, indicating that both anions and ionic liquids had influence on thermal stability of the materials.

Fig. 5 The TGA thermograms of ILs polymer materials M₁, M₂, M₃ and M₄.

Batch experiment

Effect of pH on BHb absorption of ILs polymer materials. The influence of pH on BHb absorption property of ILs polymer materials is shown in Fig. 6. It can be seen that the absorption capacity of ILs polymers changed substantially in the pH range of 5–9. The absorption capacity increased with increasing pH value and reached the maximum at pH 6.8, after which it decreased. pH 6.8 is near to the isoelectric point (pI) of BHb. On one hand, the solubility of protein and its absorption energy was at the minimum level at pI. On the other hand, electrostatic interaction was almost the minimum at pI. As a result, the following experiments were all carried out at pH 6.8.

Fig. 6 The effect of pH on BHb absorption capacity of ILs polymer materials M₁ and M₄. Absorption conditions: time = 12 h, m = 5.0 mg, V = 5.0 mL, C₀ = 1.0 mg mL⁻¹, (PBS, 10 mM). All values are means of three measurements.

Absorption kinetics. The absorption kinetics curves of ILs polymer materials for BHb are given in Fig. 7. M₁ showed a rapidly increasing absorption capacity with time. In the first two hours, the absorption capacity reached up to 88.3% of the final equilibrium capacity. In contrast, M₄ had a relatively slow absorption capacity in the first two hours. The absorption progress of two ILs polymer materials nearly reached the equilibrium in ten hours. Shi et al.²⁸ prepared an affinity membrane with chitosan and anodic aluminum oxide (AAO) to recover BHb from hemoglobin–phosphate solution. The result showed that the absorption progress achieved equilibrium in ten hours. In general, BHb molecules were first absorbed on the surface of the polymer materials at a high absorption rate in the first part of absorption kinetic plot. In the first part, a large amount of reaction sites on the surface of ILs polymer materials was accessible for BHb to combine quickly. With the time lapsing, the absorption rate was progressively low until absorption process reached equilibrium. The reaction sites were finally filled up with proteins.

Fig. 7 Absorption kinetic of BHb on ILs polymer materials M₁ and M₄. Absorption conditions: m = 5.0 mg, V = 5.0 mL, C₀ = 1.0 mg mL⁻¹, (PBS, 10 mM, pH = 6.8). All values are means of three measurements.

Absorption isotherm. The equilibrium absorption isotherms of BHb on ILs polymer materials are illustrated in Fig. 8. It can be seen that the absorption capacity increased with increasing protein concentration. The ILs polymer materials exhibited the high adsorption rate and the saturation capacity for BHb at the initial concentration of 1.0 mg mL⁻¹. The reaction sites were combined by BHb at low concentrations. The reaction sites decreased with the concentration of BHb increasing. The results illustrated that the initial concentration of BHb played an important role in the absorption experiment.

Fig. 8 Absorption isotherms of BHb on ILs polymer materials. Absorption conditions: time = 12 h, m = 5.0 mg, V = 5.0 mL, C₀ = 1.0 mg mL⁻¹, (PBS, 10 mM, pH = 6.8). All values are means of three measurements.

Absorption selectivity. In order to investigate the selectivity of the ILs polymer materials M₁ and M₄ as new protein absorbents, four typical proteins, BHb, BSA, Lyz and Cyt C, with different molecule weights, molecule sizes and pI were chosen in the present study. The experiment was carried out at the concentration of 1.0 mg mL⁻¹ and pH 6.8. The absorption capacities of the ILs polymer materials for the above proteins are showed in Fig. 9. The adsorption capacity of M₁ for BHb was 828.5 mg g⁻¹ and M₄ for BSA was 804.7 mg g⁻¹ which were much higher than the other materials. For example, T.S. Anirudhan et al.²⁹ found the absorption capacity for Cyt C of cationic Langmuir monolayers of sulfonated poly (glycidylmethacrylate)-grafted cellulose was 119.6 mg g⁻¹. Wei et al.³⁰ showed the absorption capacity of the Fe₃O₄@grapheme oxide core–shell magnetic particles for BSA reached 181.8 mg g⁻¹. Dai et al.⁴ found the absorption capacity of porous dextran microspheres for BSA was 138.9 mg g⁻¹. Yuan et al.¹⁷ prepared a polymer material with [VBIM][Cl] and the maximum absorption capacity for Lyz reached 755.1 mg g⁻¹. The protein absorption capacities of ILs polymer materials varied with the types of the ILs. The absorption capacities of two polymer materials for BHb and BSA were much higher than other proteins. The interaction between polymer materials and BSA or BHb was stronger than that between polymer materials and Lyz or Cyt C. In fact, BHb and BSA have approximately the same molecular weight and dimensions, but much larger than Lyz and Cyt C. Besides, [ABIM][Cl] polymer material showed larger absorption capacity for BHb than [VOIM][Br] polymer material, while the result was opposite for BSA. As a result, different proteins can be absorbed and separated by changing the cations of ILs. From these results, it can be inferred that the absorption of proteins was driven not only by hydrophobic interaction and size-exclusion effects, but also by multiply interactions such as electrostatic interaction, salting-out effect, hydrogen bonds.

Fig. 9 Selective absorption of BHb, BSA, Lyz and Cyt C onto the ILs polymer materials M₁ and M₄. Absorption conditions: time = 12 h, m = 5.0 mg, V = 5.0 mL, C₀ = 1.0 mg mL⁻¹,(PBS, 10 mM, pH = 6.8). All values are means of three measurements.

The competitive absorption experiments were carried out to determine the selectivity of the ILs polymer materials M₁ and M₄. Lyz-BSA mixture with large difference in pI (pI_BSA = 4.8, pI_lyz = 11.2) was selected as a binary solution for competitive absorption experiment. Besides, a ternary solution of BSA, Lyz and Cyt C, and a quaternary solution of BHb, BSA, Lyz and Cyt C were tested in this research. The results are shown in Fig. 10(a) and (b). The absorption amount of the two ILs polymer materials was lower for Lyz than for BSA. The absorption amount of the polymer materials was lower for Lyz and BSA than for only Lyz or BSA. We speculated that the results observed for M₁ could be associated with the fact that Lyz and BSA were positively and negatively charged at pH 6.8, respectively. The electrostatic interaction between imidazolium cations of the ILs polymer materials and proteins played a predominant role in the binary absorption experiment. The absorption capacity of Lyz was decreased due to electrostatic repulsion while the absorption amount of BSA was increased due to electrostatic attraction. Hence, the absorption amount of Lyz in the protein mixture solutions containing BSA was lower than that of protein mixture without BSA, and the amount of absorbed BSA was increased in the presence of Lyz. BSA has a molecular weight larger than Lyz, but have a higher absorption capacity than the latter. Moreover, the molecular size of BSA (5.0 × 7.0 × 7.0 nm) was much larger than Lyz (3.0 × 3.0 × 4.5 nm). We concluded that the size-exclusion effect was not the unique factor in this research.

Fig. 10 Competitive absorption experiment of Lyz, BSA, Cyt C and BHb onto (a) [ABIM][Cl] polymer material and (b) [VOIM][Br] polymer material. Absorption conditions: time = 12 h, m = 5.0 mg, V = 5.0 mL, (PBS, 10 mM, pH = 6.8). All values are means of three measurements.

Cyt C, which was similar to Lyz in molecular weight, molecular size and pI was tested in the ternary experiment. The differences of the absorption capacities for Cyt C in the ternary absorption experiment or batch experiment were not obvious. We speculated that the interaction between proteins did not have much effect on the absorption of Cyt C.

In the quaternary solution, the adsorption amount for BHb and BSA was much larger than other proteins onto the ILs polymer materials M₁ and M₄, respectively. The results indicated that the electrostatic interaction, hydrophobic interaction, hydrogen bonds, ionic strength may coexist between proteins and the ILs polymer materials. It can be concluded that the [ABIM][Cl] polymer material could be used preferably for the separation and absorption of BHb. BSA could be separated and absorbed by the [VOIM][Br] polymer material.

The two polymers M₁ and M₄ had high absorption capacity for BHb and BSA, respectively. The repeatability of the both polymers M₁ and M₄ was studied. The relative standard deviations (RSD) were 2.02% (n = 6) and 1.87% (n = 6) for M₁ and M₄, respectively, demonstrating a good stability of the two polymers.

Desorption experiment. In order to validate the practicability of the resulted materials, desorption experiment was carried out by two elution steps. The result showed that the desorption ratio was 63.3% and 20.6% for BSA and BHb, respectively, using PBS (10 mM, pH = 6.8). The desorption ratio reached 24.4% and 50.3% after elution with acetonitrile/water (70/30, v/v) containing 0.1% (v/v) TFA.

Conclusions

In this study, ILs polymer materials were synthesized by a polymerization reaction of AAm and [ABIM][Cl] or [VOIM][Br] in aqueous solution. The results showed a maximum absorption of 828.5 mg g⁻¹ for BHb on [ABIM][Cl] polymer material, and 804.7 mg g⁻¹ for BSA on [VOIM][Br] polymer material. The results showed that different proteins could be adsorbed and separated preferably by different ILs polymer materials. The mechanism for protein absorption on ILs polymer materials was complicated. The high protein absorption capacities of ILs polymer materials suggested that the polymer materials were promising and attractive to apply in biotechnology, sensors and biomolecular separation.

Abbreviations

[ABIM][Cl]	1-Allyl-3-butylimidazolium chloride
[VOIM][Br]	1-Vinyl-3-octylimidazolium bromide
AAm	Arcylamide
BSA	Bovine serum albumin
BHb	Bovine hemoglobin
Lyz	Lysozyme
DNA	Deoxyribonucleic acid
Cyt C	Cytochrome C
ILs	Ionic liquids
SEM	Scanning electron microscope
FT-IR	Fourier transform infrared
DSC	Differential scanning calorimeter
TGA	Thermogravimetric analysis
MBA	N,N′-methylene bisacrylamide
BHb	Bovine hemoglobin
MW	Molecular weight
BSA	Bovine serum albumin
SDS	Sodium dodecyl sulfate
APS	Ammonium persulfate
PBS	Protein buffer solution
TEMED	Tetramethylethylenediamine
UV-VIS	Ultraviolet visible
HPLC	High performance liquid chromatography
UV	Ultraviolet
TFA	Trifluoroacetic acid
AcOH	Acetic acid
PAAm	Poly-acrylamide
pI	Isoelectric point
AAO	Anodic aluminum oxide
XPS	X-ray photoelectron spectroscopy

Acknowledgements

The authors are grateful to financial support from the Ministry of Science and Technology of China (Project no. 2013AA102202), the National Natural Science Foundation of China (21375094), Tianjin Municipal Science and Technology Commission (Project no. 11ZCGHHZ01100), Special Fund for Agro-scientific Research in the Public Interest, China (No. 201203069).

Notes and references

1. S. M. Waziri, B. F. Abu-Sharkh and S. A. Ali, Biotechnol. Prog., 2004, 20, 526–532.
2. J. Li, X. P. Liao, Q. X. Zhang and B. Shi, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2013, 928, 131–138.
3. N. Welsch, L. Yan and J. Dzubiella, Polymer, 2013, 3, 1–15.
4. L. Zhu, X. Y. Liu, T. Chen, Z. G. Xu, W. F. Yan and H. X. Zhang, Appl. Surf. Sci., 2012, 258, 7126–7134.
5. C. Dai, Y. Wang and X. Hou, Carbohydr. Polym., 2012, 87(3), 2338–2343.
6. R. A. Sheldon, R. M. Lau, M. J. Sorgedrager, F. V. Rantwijk and K. R. Seddon, Green Chem., 2002, 4, 147–151.
7. D. R. MacFarlane, M. Forsyth, P. C. Howlett, J. M. Pringle, J. Sun, G. Annat, W. Neil and E. I. Izgorodina, Acc. Chem. Res., 2007, 40(11), 1165–1173.
8. S. Pandey, Anal. Chim. Acta, 2006, 556, 38–45.
9. Y. C. Pei, J. J. Wang, X. P. Xuan, J. Fan and M. H. Fan, Environ. Sci. Technol., 2007, 41(14), 5090–5095.
10. X. X. Han and W. A. Daniel, Acc. Chem. Res., 2007, 40(11), 1079–1086.
11. A. J. Carmichael and K. R. Seddon, J. Phys. Org. Chem., 2000, 13, 591–595.
12. K. Shimojo, N. Kamiya, F. Tani, H. Naganawa, Y. Naruta and M. Goto, Anal. Chem., 2006, 78(22), 7735–7742.
13. J. H. Wang, D. H. Cheng, X. W. Chen, Z. Du and Z. L. Fang, Anal. Chem., 2007, 79(2), 620–625.
14. Y. C. Pei, J. J. Wang, K. Wu, X. P. Xuan and X. J. Lu, Sep. Purif. Technol., 2009, 64, 288–295.
15. L. Vidal, M. L. Riekkola and A. Canals, Anal. Chim. Acta, 2012, 715, 19–41.
16. R. Marcilla, J. A. Blazquez, J. Rodriguez, J. A. Pomposo and D. Mecerreyes, Polym. Chem., 2004, 42, 208–212.
17. S. F. Yuan, Q. L. Deng, G. Z. Fang, M. F. Pan, X. R. Zhai and S. Wang, J. Mater. Chem., 2012, 22, 3695–3972.
18. Y. Liu, Y. L. Yu, M. Z. Chen and X. Xiao, Chem. Eng. Technol., 2011, 2, 1–7.
19. K. E. Gutowski, G. A. Broker, H. D. Willauer, J. G. Huddleston, R. P. Swatloski, J. D. Holbrey and R. D. Rogers, J. Am. Chem. Soc., 2003, 125(22), 6632–6633.
20. C. Y. He, S. H. Li, H. W. Liu, K. Li and F. Liu, J. Chromatogr. A, 2005, 1082, 143–149.
21. M. J. Ruiz-Angel, V. Pino, S. Carda-Broch and A. Berthod, J. Chromatogr. A, 2007, 1151, 65–73.
22. S. Dreyer and U. Kragl, Biotechnol. Bioeng., 2008, 99, 1416–1424.
23. Y. Zhao, Y. Cao, Y. L. Yang and C. Wu, Macromolecules, 2003, 36(3), 855–859.
24. N. A. Peppas, P. Bures, W. Leobandung and H. Ichikawa, Eur. J. Pharm. Biopharm., 2000, 50, 27–46.
25. G. Q. Fu, H. Yu and J. Zhu, Biomaterials, 2008, 29, 2138–2142.
26. F. Hachem, B. A. Andrews and J. A. Asenjo, Enzyme Microb. Technol., 2009, 19, 507–517.
27. N. Bereli, M. Andac, G. Baydemir, R. Say, I. Y. Galaev and A. Denizli, J. Chromatogr. A, 2008, 1190, 18–26.
28. W. Shi, Y. Q. Shen, D. T. Ge, M. Q. Xue, H. H. Cao, S. Q. Huang, J. X. Wang, G. L. Zhang and F. B. Zhang, J. Membr. Sci., 2008, 325, 801–808.
29. T. S. Anirudhan and P. Senan, Chem. Eng. J., 2011, 168, 678–690.
30. H. Wei, W. S. Yang, Q. Xi and X. Chen, Mater. Lett., 2012, 82, 224–226.

---