Anti-nonspecific adsorption segments-assisted self-driven surface imprinted fibers for efficient protein separation
Zuoting Yang 1, 2, Ting Wang 1, Yabin Wang 1, 3, Qiuyu Zhang 1, 211* Corresponding authors:, Baoliang Zhang1, 4 22Northwestern Polytechnical University, Youyi Road 127#, Xi’an (710072), China. Email: qyzhang@nwpu.edu.cn, blzhang@nwpu.edu.cn
1 School of Chemistry and Chemical Engineering, Northwestern Polytechnical University, Xi’an, 710129, P. R. China
2 Xi’an Key Laboratory of Functional Organic Porous Materials, Northwestern Polytechnical University, Xi’an, 710072, P. R. China
3 Shaanxi Key Laboratory of Chemical Reaction Engineering, College of Chemistry and Chemical Engineering, Yan’an University, Yan’an 716000, PR China
4 Shaanxi Engineering and Research Center for Functional Polymers on Adsorption and Separation, Sunresins New Materials Co. Ltd., Xi’an, 710072, China
Abstract: At present, the development of high-performance protein imprinted materials is still a research hotspot in the field of protein imprinting. Herein, anti-protein adsorption segment (CBMA)-assisted self-driven BSA surface imprinted fibers MTCFs@SIP@CBMA with high recognition selectivity are pioneered using the strategies of combining magnetic nanomaterial surface imprinting technique with amino-Michael addition. The special structure of the carrier MTCFs endows MTCFs@SIP@CBMA with magnetic performance and self-driven adsorption performance, which simplifies the separation process while improving the adsorption capacity and accelerating the adsorption rate. The adsorption capacity for BSA reached 395.26 mg/g within 30 min. The introduction of CBMA segments on the surface after imprinting by amino-Michael addition makes its polymer chain length and position controllable. Under the strongest anti-nonspecific adsorption effect, MTCFs@SIP@CBMA exhibit excellent specific identification to BSA from mixed proteins. Additionally, MTCFs@SIP@CBMA show considerable reusability. Therefore, MTCFs@SIP@CBMA are expected to be applied in efficient separation of proteins in biological samples.
Keywords: protein imprinted materials; magnetic tubular nanofiber; anti-protein adsorption; self-driven adsorption; high recognition selectivity
Introduction
The highly selective recognition of proteins provides important technical support for the development of protein drugs, medical diagnosis, and the exploration of the pathogenesis of major diseases based on proteomics. It is an important research topic in the fields of life sciences and biotechnology1, 2. Therefore, the separation, purification and detection of proteins are particularly important. As a new type of protein identification and separation material, protein imprinted polymers have the advantages of easy preparation, low cost, high selectivity, strong practicability and promising mechanical stability, so it is widely favored by researchers3-5. After a prolonged endeavor, in recent years, the imprinting methods for proteins and other types of macromolecular templates have made some research progress6-9. However, compared with imprinted polymers based on small molecules as templates, the recognition selectivity, separation rate and separation amount of protein imprinted polymers are difficult to meet the needs of practical applications. This is due to the large molecular volume, complex structure and flexible conformation of proteins 10-12. In view of the specificity of protein structure, researchers have developed different strategies for protein imprinting, including epitope imprinting13-15, surface imprinting16-18, the introduction of stimulus-responsive functional monomers or cross-linking agents19-21 and anti-protein adsorption segments22-24, etc.
At present, among many protein imprinting techniques, surface imprinting based on micro/nano materials is a popular and most suitable method for preparing protein imprinted polymers. Surface imprinting refers to coating an ultra-thin polymer layer containing template molecules on the surface of the micro/nano carrier materials. The imprinting sites are located near the surface of the outer shell layer, making it easier for proteins to enter and exit the recognition sites of the polymers. Therefore, it is an effective way to overcome the mass transfer difficulties caused by the large molecular size of the protein and increase the adsorption amount of the template proteins. For surface protein imprinting, the choice of carrier is crucial. Magnetic micro/nano materials can move in a controlled direction under an external magnetic field, avoiding tedious sample handling and separation processes. So, they have been widely used in many fields25-27. Based on this advantage, protein imprinting also began to develop magnetic micro/nano materials as polymeric carriers. A series of protein imprinting was carried out on their surfaces to obtain magnetic surface imprinted polymers with easy separation and good adsorption performance28-31. For example, our research group previously prepared surface imprinted magnetic graphene (Fe3O4@rGO@MIPs) microspheres32 and surface imprinted nitrogen-doped magnetic carbon nanotubes (N-MCNTs@MIPs)33 by imprinting bovine serum protein (BSA) on the surface of magnetic graphene microspheres (Fe3O4@rGO) and nitrogen doped magnetic carbon nanotubes (N-MCNTS). The two imprinted materials could achieve rapid separation under the external magnetic field, and show rapid binding ability and high adsorption capacity. The adsorption capacity of BSA was 317.58 mg/g and 150.86 mg/g, respectively. In addition, considering the complex structure of the protein and a variety of amino acid residues rich on its surface, it is inevitable that the protein will interact with the material surface outside the imprinted sites, resulting in non-specific adsorption. Consequently, the specific recognition ability of the imprinted materials is reduced. Therefore, how to suppress non-specific adsorption is also an urgent problem in the field of protein imprinting. Studies have proved that amphoteric polymers could form a hydration layer on their surface through electrostatic interaction or hydrogen bonding with water molecules, which significantly reduces the adsorption of proteins on the surface of materials34, 35. Moreover, the amphoteric polymers possess a charged positive and negative charge center, which would not change the spatial conformation of the binding proteins. The super-hydrophilic characteristics determine that the coupling protein could still maintain its own activity. Therefore, introducing amphoteric polymers with anti-protein adsorption properties into the imprinting shell is an ideal method for preparing protein imprinted materials with high specific recognition ability. For instance, Wang et al. 36 introduced sultaine methacrylate (SBMA) as an anti-protein adsorption chain into the imprinted shell, and successfully prepared BSA imprinted magnetic microspheres (Fe3O4@SiO2@MIP) by precipitation copolymerization. The imprinted microspheres showed high adsorption capacity (QMIP=116.39 mg/g) and excellent selectivity (IF=4.73) for BSA. However, most of the reported methods for introducing anti-protein adsorption segments are directly copolymerizing them with functional monomers36-38. It is difficult to ensure that all anti-protein components are not present in the imprinting sites, which is prone to reduce the amounts of imprinted sites. As a result, the adsorption capacity is reduced. Additionally, the length and arrangement of the anti-protein adsorption chain segments in the imprinted layer cannot be precisely controlled. Based on the above reasons, it is urgent to develop a new method to introduce anti-protein adsorption polymer chains. Noteworthily, the conformational changes of the proteins during the imprinting process would also cause the reduction of the selective recognition of imprinting sites. Therefore, mild experimental conditions should be selected as far as possible to prepare the protein imprinted polymers for avoiding the conformational change of proteins during the imprinting process. Dopamine (DA) could self-polymerize on the surface of the materials under dissolved oxygen and alkaline conditions, and the polymerization conditions are very mild. It has been widely used as a functional monomer for protein imprinting39-41.
Aiming at the difficulties of low adsorption capacity, slow adsorption rate, difficult mass transfer and poor selective recognition of protein imprinted materials, this work developed an anti-protein adsorption chain 3-[[2-(Methacryloxy)ethyl]dimethylammonium]propionate (CBMA)-assisted self-driven BSA surface imprinted material with high recognition selectivity (MTCFs@SIP@CBMA) by adopting the strategy of combining magnetic nanomaterial surface imprinting with amino-Michael addition. The carrier is a magnetic tubular carbon nanofiber with porous tube wall. Its special structure endows imprinted materials with magnetic properties and self-driven performance, which are beneficial to simplify the separation process, increase the adsorption capacity, and accelerate the adsorption rate. To improve the recognition selectivity of the imprinted materials, on the one hand, the protein structure-friendly biological dopamine is selected as a functional monomer to perform the coating of imprinted polymers, ensuring that the imprinting process is mild and avoiding the conformational change of proteins. On the other hand, CBMA is selected as the anti-protein adsorption chain segment to be introduced onto the imprinted polymer shell on the surface of materials. The purpose is to reduce non-specific adsorption. Different from the copolymerization method previously reported, this work adopts the method of first coating the imprinted polymers on the surface of the carriers and then grafting the anti-protein adsorption chain segments CBMA on the surface of the imprinted shell layer by amino-Michael addition. In this way, the position and length of the CBMA polymer chain are controllable, and the recognition selectivity of the imprinted materials can be improved on the premise of ensuring a high adsorption capacity. Based on the above design, the influence of the length of CBMA polymer chain on the adsorption capacity and imprinting factor of MTCFs@SIP@CBMA is investigated. In addition, the developed materials are systematically characterized, and the adsorption performance, recognition selectivity, physical separation ability and reusability of MTCFs@SIP@CBMA are simultaneously studied.
Experimental section
Synthesis of BSA surface imprinted magnetic tubular carbon nanofibers
The preparation process of BSA surface imprinted magnetic tubular carbon nanofibers (BSA-MTCFs@SIP) was divided into two steps. In the first step, the carrier magnetic tubular carbon nanofibers (MTCFs) were synthesized using the method previously reported by our research group.[42, 43] The specific preparation process was displayed in the supporting information. In the second step, the surface of MTCFs was coated with a polydopamine (PDA) imprinted shell. The typical process was as follows: 30 mg of MTCFs and 60 mg of Tween 20 were ultrasonically dispersed in 20 mL Tris -HCl buffer (pH=8.5), and 20 mg BSA was added. After stirring for 30 min, 10 mL of Tris-HCl buffer solution containing 30 mg of DA was quickly added to the above dispersion. The reaction was carried out for 14 h at room temperature. The products were magnetically separated and washed repeatedly with ultrapure water to remove unreacted monomer and template protein, BSA-MTCFs@SIP was obtained under vacuum drying.
2.2 Preparation of BSA imprinted magnetic carbon nanofibers with abundant CBMA polymer segments on the surface
CBMA polymer segments grafted BSA imprinted magnetic carbon nanofibers (BSA-MTCFs@SIP@CBMA) were obtained by one-step reaction of amino-Michael addition. Firstly, 30 mg of BSA-MTCFs@SIP was dispersed ultrasonically in a three-mouth flask containing 40 mL water and 10 mL ethanol. Then, an aqueous solution with 150 mg CBMA was dropped into the above dispersion, followed by the addition of 0.375 mL triethylamine. After the reaction was performed at 25°C for 24 h, the products were collected by magnetic separation, washed repeatedly with ultrapure water, and dried in vacuum to obtain BSA-MTCFs@SIP@CBMA.
2.3 Elution of BSA-MTCFs@SIP@CBMA
A certain amount of BSA-MTCFs@SIP@CBMA was dispersed in 6% (V/V) glacial acetic acid solution and washed at 25℃ to remove template molecules. The absorbance of BSA in the eluent was tracked and measured at 278 nm by UV-vis spectrophotometer until there was no obvious absorption peak in the eluent. After washing with ultrapure water to neutral, vacuum drying was performed to obtain CBMA grafted surface imprinted magnetic tubular carbon nanofibers (MTCFs@SIP@CBMA). As a control, CBMA grafted non-imprinted magnetic carbon nanofibers (MTCFs@NIP@CBMA) were prepared using a method similar to that of MTCFs@SIP@CBMA, but without the addition of template molecule BSA.
Results and discussion
Design and synthesis of MTCFs@SIP@CBMA
In order to construct high-performance protein imprinted materials, a zwitterionic polymer CBMA-assisted self-driven BSA surface imprinted magnetic tubular carbon nanofibers MTCFs@SIP@CBMA was designed and synthesized in this work. The preparation process is designed in Figure 1. Firstly, magnetic tubular carbon nanofibers (MTCFs) were prepared by vacuum high temperature calcination using polymer nanofibers without extracting the template FeCl3 as precursor. On the one hand, MTCFs were magnetic, simplifying the separation process without the need for cumbersome, energy-consuming techniques such as filtration or centrifugation. On the other hand, the tube wall of MTCFs possessed abundant pores and contained internal cavities. This special structure gave the imprinted materials with self-driven adsorption performance. When it was dispersed in a high-concentration BSA solution, a concentration gradient was formed between the cavity inside the tube and the outside of the tube, resulting in an osmotic pressure difference. The osmotic pressure difference would drive BSA molecules to spontaneously bind to the imprinted sites on the surface, and further migrated to the internal cavity through the pores of the tube wall. Accordingly, the adsorption capacity was significantly increased and the adsorption speed was accelerated. Therefore, MTFs was chosen as the carrier to cover the imprinting layer. Utilizing BSA as template molecules and biofriendly dopamine as functional monomers, BSA-MTCFs@SIP were prepared by coating imprinted polymers on the surface of MTCFs through oxidative self-polymerization. Subsequently, to improve the recognition selectivity of the imprinted materials, the zwitterionic polymer CBMA as an anti-protein adsorption segment was grafted on the surface of the imprinting layer to reduce the non-specific adsorption of materials. Different from the copolymerization method previously reported, in this manuscript, the anti-protein adsorption chain segments were introduced by first coating the polydopamine imprinted layer on the carrier and then grafting CBMA on the surface of the imprinted layer via amino-Michael addition. Therefore, the location and length of CBMA were controllable, which can improve the recognition selectivity of imprinted materials under the premise of ensuring high adsorption capacity. After the elution step by 6% HAc, the surface of MTCFs@SIP@CBMA produced imprinting sites with high recognition selectivity that match BSA in three-dimensional space and functional groups.