Preparation of bovine serum albumin imprinting sensitive hydrogels using ionic liquid as co-monomer and stabilizer
Graphical abstract
A polymerizable and biocompatible ionic liquid is designed and used as co-monomer and stabilizer in preparation of bovine serum albumin imprinted hydrogels.
Introduction
Molecular imprinting is a technique to create artificial receptors by copolymerizing functional monomers and crosslinkers in the presence of a template molecule. In the mixture of pre-polymer, functional monomers interact with a template in a certain spatial arrangement by hydrogen bonding, electrostatic or hydrophobic interactions. After the polymerization of the monomers and crosslinkers, the spatial arrangement around the template is fixed. The removal of the template leaves the complementary binding sites with specific recognition ability. Although the first study on molecular imprinted polymer (MIP) was published 80 years ago [1], the prosperous development of molecular imprinting began only in the 1980s and it has been widely applied in sensor, catalysis, chromatographic separation and solid phase extraction (SPE) field. Haupt et al. reported a versatile fiber-optic fluorescence sensor based on MIP for herbicide [2], and its sensitivity was significantly improved by adding a signal enhanced material. Their ingenious method of using a signaling monomer in MIP has extensively simplified the detection procedure. Guo has demonstrated a new strategy of reactant–product-dual-template imprinted capsule for the simultaneous degradation and elimination of pesticide [3]. The novel strategy can effectively enhance the catalytic efficiency of the molecular imprinting catalyst by simply combining the characteristics of the imprinting reactant and product, and could be widely used in environmental fields. Manesiotis et al. reported the preparation of MIPs against S-ibuprofen [4], which was used as the stationary phase, for the complete resolution of racemic ibuprofen in predominantly aqueous mobile phases. The SPE of ibuprofen from tablets, using MIP as sorbents, resulted in 92.2% recovery.
Low molecular weight MIPs have been successfully used for imprinting [5], [6], [7]. However, the imprinting of larger and more complex molecules, such as proteins, enzyme, DNA and viruses, has been very difficult [8]. In a highly crosslinked MIP, the removal of the template is a very important issue for both micro- and macromolecules. However, macromolecules are more difficult to remove and transport compared to micromolecule in the tough MIP matrix. Furthermore, the imprinting process must be carried out in a stable and gentle aqueous environment because of the sensitive structural nature of biomacromolecules. Moreover, hydrogen bonding interaction is usually applied in organic solvents to obtain the good imprinted effect, but the situation is inapplicable in aqueous phase. Therefore, biomarcromolecules imprinting technology needs to be modified to overcome the above mentioned difficulties.
Sensitive hydrogels are those which can reversibly change their dimension by altering external environmental conditions, such as temperature, ionic strength, pH, etc. Due to their large volume change, sensitive hydrogels can be advantageously applied in macromolecule imprinting. Until now, several studies have been reported on biomacromolecule imprinted sensitive hydrogel [9], [10], especially for imprinting proteins. Hua et al. utilized N-[3-(dimethylamino) propyl]-methacrylamide as the charged functional monomer and NIPA as the sensitive assistant monomer to prepare a stimuli-responsive protein imprinted hydrogel [11], and their template protein could be easily removed by controlling ionic strength. They also reported the preparation of MIH based on NIPA monomer with specific protein sensitivity compared to NIH [12]. Ran et al. reported a new method for the preparation of sensitive MIH based on NIPA at two different temperatures (25 and −20 °C) [13]. The results showed that MIH produced at −20 °C, exhibited good structural regularity and adsorption selectivity. Although there has been significant development in MIH for biomacromolecular imprinting, the focus on the structural integrity and stability of the protein in the pre-polymerization system has not been studied yet. Even though the buffer solutions have been used as the stabilizing reagent for the template, the protein could be damaged by the strong interaction with functional monomers in pre-polymerization and polymerization processes. Therefore, for biomacromolecule imprinting, it is necessary to maintain the protein nature unfolding structure and stability in a chemical reaction by monitoring its configuration state and preferred pathways for the stabilization.
Ionic liquids (ILs) refer to the low temperature-melting organic salts that are typically composed of bulky organic cations and charge diffuse inorganic or organic anions [14]. ILs have many favorable properties including negligible vapor pressure, good thermal stability, a wide liquid range, low flammability, powerful dissolution ability, high ionic conductivity and designability. Thus, ILs have been widely used as the solvent, template [15], catalyst and reactive monomer in the fields of asymmetric synthesis [16], [17], adsorbing material [18], [19], [20], fluorescence modified material [21], polymerization [22], [23] and biomaterial separation [24], [25], [26]. Recently, ILs used for stabilizing and activating enzymes and proteins have drawn significant attention [27], [28], [29]. This study is focused on the stabilizing effect of ILs on the proteins and enzymes in accordance with the Hofmeister series [30], which reflect the ion-induced modifications of water's hydrogen (H)-bonded network [31]. The series of ions ranked by the protein stabilizing efficiency is expressed as follows [32]:
The double bar indicates the crossover from stabilizing to destabilizing behavior. In the above series, the large ions of low charge density defined as chaotropes, which break the H-bond bulk structure of water, and the small ions of high charge density defined as kosmotropes, which were believed to enhance the H-bonded bulk structure of water [33]. The best stabilizing effect on protein was accomplished by the ionic combination of a chaotropic cation with a kosmotropic anion. Taking advantage of this series, Fujita et al. “designed” a biocompatible IL, choline dihydrogen phosphate ([chol][dhp]), to enhance the stability of cytochrome c and metallo protein [34], [35]. Therefore, it is reasonable to utilize the Hofmeister effect to design an IL for the protein stabilization.
In this study, we present the design and synthesis of a novel IL named 1-vinyl-3-aminoformylmethyl imidazolium chloride ([VAFMIM]Cl). The [VAFMIM]Cl consisted of a kosmotropic anion and a short alkyl imidazolium cation, which might act as a chaotropic cation [36], to afford good biocompatibility. Moreover, the electrostatic force generated by the imidazolium ring, and the hydrogen bond induced by the amide group would also interact with the template protein. Therefore, the [VAFMIM]Cl was not only used as a stabilizer, but also a co-monomer in MIH. NIPA was chosen as the assistant monomer providing the polymer “swelling/shrinking” controlled ability in the elution process. The protocol for the synthesis of MIH is shown in Scheme 1. The recognition ability of the resultant MIHs and NIHs were conducted in various adsorption tests and their selectivity were also investigated.
Section snippets
Materials
N-isopropylacrylamide (NIPA) was purchased from Acros Organics N,N-methylenebisacrylamide (BIS), acrylamide (AAm), ammonium persulfate (APS), and N,N,Nʹ,Nʹ-tetramethylethylenediamine (TEMED) were obtained from Sigma. 1-Vinylimidazole and 2-chloroacetamide were purchased from Alfa Aesar. Tris(hydroxymethy)aminomethane (Tris) was purchased from J.T. Baker (Phillipsburg, NJ). 2-(dimethylamino)ethylmethacrylate (DMAEMA), bovine calf serum(BCS), bovine serum albumin (BSA; MW 66.4 kDa, pI 4.8),
Results and discussion
The hydrogels based on NIPA monomer can undergo a reversible volume transition between shrinking and swelling, which are controlled by external temperature. Fig. 1 shows the effect of temperature on the degree of swelling of the MIH and NIH. For the temperature responsive MIH and NIH, the degree of swelling drastically decreased with increasing temperature above the lower critical solution temperature (LCST). Although the LCST of PNIPA was about 32 °C [39], the LCST of both the MIH and NIH
Conclusion
In this study, a novel biocompatible IL was designed and used as co-monomer and protein stabilizing reagent for the preparation of sensitive BSA imprinted hydrogel. The biocompatible IL has been proved to significantly enhance the stability of BSA in Tris–HCl buffer. The preparation and elution conditions of the MIHs were optimized to afford the best absorption capacity and selectivity. The optimized MIH were subsequently used in the adsorption dynamic, adsorption isotherm, adsorption
Acknowledgment
The authors greatly appreciate the financial supports by the National Natural Science Foundation of China (NO.21174111).
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