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.