A sol-gel derived pH-responsive bovine serum albumin molecularly imprinted poly(ionic liquids) on the surface of multiwall carbon nanotubes

Highlights

• An alkyl-functionalized ionic liquid was used as monomer, stabilizer and catalyst to prepare MIPs.

• The MWCNTs@BSA-MIPIL was prepared via a multi-step sol-gel route.

• The imprinting conditions were optimized by investigating molecular interactions between templates and monomers.

• The MWCNTs@BSA-MIPIL was found to be pH-responsive.

• The MWCNTs@BSA-MIPIL demonstrated high adsorption capacity, good imprinting effect and strong shape selectivity to BSA.

Abstract

A pH-responsive surface molecularly imprinted poly(ionic liquids) (MIPILs) was prepared on the surface of multiwall carbon nanotubes (MWCNTs) by a sol-gel technique. The material was synthesized using a 3-aminopropyl triethoxysilane modified multiwall carbon nanotube (MWCNT-APTES) as the substrate, bovine serum albumin (BSA) as the template molecule, an alkoxy-functionalized IL 1-(3-trimethoxysilyl propyl)-3-methyl imidazolium chloride ([TMSPMIM]Cl) as both the functional monomer and the sol-gel catalyst, and tetraethoxysilane (TEOS) as the crosslinking agent. The molecular interaction between BSA and [TMSPMIM]Cl was quantitatively evaluated by UV–vis spectroscopy prior to polymerization so as to identify an optimal template/monomer ratio and the most suitable pH value for the preparation of the MWCNTs@BSA-MIPILs. This strategy was found to be effective to overcome the problems of trial-and-error protocol in molecular imprinting. The optimum synthesis conditions were as follows: template/monomer ratio 7:20, crosslinking agent content 2.0–2.5 mL, temperature 4 °C and pH 8.9 Tris–HCl buffer. The influence of incubation pH on adsorption was also studied. The result showed that the imprinting effect and selectivity improved significantly with increasing incubation pH from 7.7 to 9.9. This is mainly because the non-specific binding from electrostatic and hydrogen bonding interactions decreased greatly with the increase of pH value, which made the specific binding affinity from shape selectivity strengthened instead. The polymers synthesized under the optimal conditions were then characterized by BET surface area measurement, FTIR, thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The adsorption capacity, imprinting effect, selective recognition and reusability were also evaluated. The as-prepared MWCNTs@BSA-MIPILs were also found to have a number of advantages including high surface area (134.2 m² g⁻¹), high adsorption capacity (55.52 mg g⁻¹), excellent imprinting effect (imprinting factor of up to 5.84), strong selectivity (selectivity factor of 2.61 and 5.63 for human serum albumin and bovine hemoglobin, respectively), and good reusability.

Introduction

Molecular imprinting (MI) is a versatile approach for the creation of synthetic polymers, namely molecularly imprinted polymers (MIPs) with tailored recognition sites for a given target or group of target molecule [1], [2]. As a new class of synthetic receptors, MIPs have attracted considerable attention in the field of clinical analysis [3], medical diagnostics [4], environmental monitoring [5], and enzyme mimicking catalysis [6], thanks to the desired predetermination, specific recognition and wide practicability. To date, the imprinting of small molecules has been well established with the non-covalent and covalent methods formulated by Vlatakis and Wulff [1], [2]. However, creating MIPs that recognize biomacromolecules such as proteins still faces serious challenges. There are a number of reasons for this including the large molecular size, complex structure, flexible conformation, potential for denaturation, water compatibility issues, and multiple binding sites with the functional monomers [7], [8], [9].

Surface molecular imprinting, grafting a MIP layer on the surface of a solid substrate (e.g. silica particle, carbon nanomaterial, polymer support and magnetic nanoparticle), is one of the more promising alternatives for biomacromolecules [10], [11], [12]. It can, to some extent, overcome not only the problems that are inherent by the nature of biomacromolecules but also the drawbacks resulting from traditional embedding methods. These surface imprinted polymers possess certain advantages, such as low steric hindrance, strong mechanical stability and high loading of imprinting cavities, leading to acceleration in mass transfer and possibly an increase in the imprinting effect [13], [14]. Multiwalled carbon nanotubes (MWCNTs), due to their high mechanical strength, large specific surface area, excellent chemical stability, and flexible surface modification property, have good potential to serve as cores or substrates when fabricating core-shell surface-imprinted polymer nanotubes or nanowires [15], [16]. For example, a surface imprinted polydopamine film was developed for specific recognition of papain by Liu et al. [17], using MWCNTs as the substrates, papain as the template molecules, and dopamine as the functional monomers. The polymer layer deposited on the surface of MWCNTs was found to be as thin as 15–20 nm, which made the recognition sites more accessible for the template molecules. Therefore, the papain molecules could reach the imprinting sites more freely and took less time to reach adsorption equilibrium (within 35 min). The developed MWCNTs@MIPs also possessed excellent binding capacity and selectivity for papain molecules due to high loading of imprinting sites on the surface of the thin polymer layer. MWCNTs, due to their good potential for surface modification, can also be covalently or noncovalently modified by Fe₃O₄ [18], Fe₃O₄@chitosam [19], Fe₃O₄@SiO₂ [20], and quantum dots (QDs) [21], etc, in order to improve their performance as substrate materials in different application field. For instance, Ding et al. [21] designed a fluorescent biomimetic sensor for specific recognition of bovine serum albumin (BSA) by creating a MIP layer on the surface of MWCNTs-QDs. The results showed that the rebinding of BSA to the imprinting sites significantly quenched the luminescence of the MIP-coated MWCNTs-ODs, and the quenching constant for BSA was much higher than bovine hemoglobin (BHb) and lysozyme (Lys). Moreover, the template selectivity, fluorescence stability and mass transfer efficiency of the MIP-coated MWCNTs-ODs were also superior to the MIP-coated ODs due to the introduction of MWCNTs with large specific surface areas.

A key factor affecting the imprinting effect, selectivity and binding capacity of the MIP is the stability of the formed template-monomer complex. The functional monomer that can interact most intensively with the template molecule will give the template-monomer complex the highest stability. This will facilitate subsequent formation of specific recognition sites that are complementary to the template molecules in size, shape and functionality. Ionic liquids (ILs), a new class of solvent entirely composed of ions, have many unique properties including negligible vapor pressure, high thermal stability, good film-forming ability, and good solvating properties [22], [23]. They can also provide multiple interactions, such as electrostatic, hydrogen bonding, π-π and hydrophobic interactions with various organic compounds and biomacromolecules [24], [25]. The use of ILs for stabilizing and activating enzymes and proteins has gained significant attention, as well [26], [27], [28], [29]. These unique characteristics have made ILs desirable extraction media and selective stationary phases in the area of extraction or separation [30], [31]. In the molecular imprinting field, they have been investigated not only as solvents and porogens [32], [33] but also as stabilizers [34] and functional monomers [35], [36]. The protein imprinted polymer based on IL functional monomer was first reported by Yuan et al. [37], in which, 1-vinyl-3-butylimidazolium chloride was used as monomer, acrylamide as co-monomer, Lys as template molecule, and MWCNT as the substrate. The results showed that the introduction of 1-vinyl-3-butylimidazolium chloride IL as monomer resulted in an increase in imprinting factor in comparison with the introduction of vinyl imidazole monomer. A molecularly imprinted electrochemical sensor for BSA recognition was fabricated by Wang and his coworkers [38] via in-situ electrochemical polymerization of 3-(3-aminopropyl)-1-vinylimidazolium tetrafluoroborate IL on the surface of a MWCNT modified glassy carbon electrode. The molecularly imprinted film electrode was found to have enhanced accessibility, good imprinting effect and high specificity towards BSA. They also exhibited good sensing performance to BSA with low detection limit and wide linear range. Qian et al. [34] developed a sensitive BSA imprinted N-isopropylacrylamide (NIPAM) hydrogel with the help of 1-vinyl-3-aminoformylmethyl imidazolium chloride ([VAFMIM]Cl) IL as co-monomer and stabilizer. The stabilizing effect of the IL on BSA was verified by circular dichroism. The introduction of IL to the prepolymerization solution also improved the imprinting effect and specific recognition ability of the hydrogel to BSA. A BSA imprinted hydrogel prepared with equimolar ratio of 2-(dimethylamino)ethylmethacrylate (DMAEMA) was also prepared as the reference in order to compare the stabilizing effect and recognition ability of the IL with conventional monomer. The results showed that hydrogel composed of IL exhibited higher adsorption capacity and imprinting effect than that comprised of conventional DMAEMA. This is mainly because [VAFMIM]Cl IL, as a biocompatible and stabilizing reagent, maintained the structure of template protein during the polymerization reaction. It is also attributed to the strong electrostatic interaction caused by the imidazolium cation, and hydrogen bonding interaction induced by the aminoformyl group in [VAFMIM]Cl. A Lys-imprinted core-shell microsphere was also successfully synthesized by Qian et al. [39] using hydroxyethyl acrylate as monomer and 5% choline dihydrogen phosphate IL as thermal stabilizer via thermal initiation at 75 °C. The result obtained from circular dichroism and activity assay indicated that the structure and activity of template protein was maintained very well even under such high reaction temperature. The imprinting factor and selectivity factor of the Lys-imprinted microsphere synthesized with ILs as thermal stabilizer was much better in comparison with those prepared without ILs, indicating a strong relationship between protein conformation and the specific recognition ability.

The recognition of protein molecules by MIPs is believed to be a combination of shape selectivity by the imprinting cavities and multiple interactions by the complementary functionalities in monomers [40], [41]. Generally, the formed imprinting sites around the monomers lead to a specific binding with the target proteins, while the randomly distributed monomers in the polymers result in a non-specific adsorption with both the templates and the competitive proteins. Therefore, an appropriate ratio of template/monomer should be introduced to the polymerization system to maximize the formation of effective imprinting sites while minimize the production of non-specific interactions. The interactions between templates and monomers are greatly influenced by the temperature, pH and ionic strength of the external environments. Therefore, these environmental parameters should also be optimized during the synthesis of the MIPs in order to increase the stability of the template-monomer complex [9], [42]. Although the synthesis procedure of the MIPs is relatively simple and inexpensive, the acquisition of the best conditions for polymer preparation is not trivial in practice. It is commonly based on a trial-and-error approach, which is tedious and reagent consuming. Moreover, the optimization is often limited to one parameter at a time, which overlooks the interplay of different parameters. In order to overcome the problems of trial-and-error imprinting protocol, some more systematic strategies, such as the chemometrics approaches [43], [44], [45], combinatorial synthesis [46], and high throughput screening [47] are reported in recent researches. However, these methods also suffer from several drawbacks. For example, it is difficult to conduct the thermodynamic calculations on a multi-component system like MIP. The molecular interaction between templates and monomers can be investigated by ultraviolet–visible spectroscopy (UV–vis) [48], ¹H nuclear magnetic resonance (¹H NMR) [49], [50], Fourier transform infrared spectroscopy (FTIR) [51] and isothermal titration calorimetry [52], which are relatively easy to carry out compared to the systematic strategies mentioned above. These studies can provide reliable information to help us find the best functional monomers, the optimum template/monomer ratio, and the most suitable external environmental parameters [53].

Although MIPs based on IL monomers have shown some advantages, the research in this area is still quite limited and inadequate. To date, only a few ILs have been investigated as monomers for the preparation of MIPs for protein recognition despite thousands of ILs have been discovered in the past decades [34], [37], [38], [39]. Moreover, all of these IL-based MIPs are prepared via free radical polymerization reaction. What's more, the recognition mechanism between ILs and template proteins is still unclear, and need to be clearly clarified. Recently, sol-gel technique has been extended to the field of protein molecular imprinting, due to its good compatibility with protein molecules and aqueous environments as well as mild polymerization conditions (i.e. adjustable pH and low temperature) [54], [55], [56]. However, there are no relevant studies on the development of protein imprinted IL-based polymers by sol-gel technique even if IL can be easily modified with an alkoxy functional group. In this work, a molecularly imprinted polymeric ionic liquid (MIPIL) film was prepared on the surface of multiwall carbon nanotubes (MWCNTs) using a sol-gel technique with BSA as the template, alkoxy-functionalized IL 1-(3-trimethoxysilyl propyl)-3-methyl imidazolium chloride ([TMSPMIM]Cl) as the monomer, 3-aminopropyl triethoxysilane modified MWCNTs (MWCNTs-APTES) as the substrate, and tetraethoxysilane (TEOS) as the crosslinking agent. The hydrolysis and polycondensation process of the sol-gel reaction was optimized. The molecular interaction between BSA and [TMSPMIM]Cl was quantitatively evaluated by UV–vis spectroscopy before polymer synthesis so as to find an optimal template/monomer ratio and a suitable pH for the preparation of the MWCNTs@BSA-MIPILs. The content of the crosslinking agent was also optimized. The responsivity of the as-prepared MWCNTs@BSA-MIPILs to external stimuli, such as pH of the incubation solution was also investigated. The polymers synthesized under the optimal conditions were characterized by BET surface area measurement, FTIR, thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The adsorption thermodynamics, selectivity and reusability were also evaluated in detail.