Protein-imprinted Polymers Research Articles

Advances in the selection of functional monomers for molecularly imprinted polymers: A review.

Molecularly imprinted polymers, a type of special polymer materials, are widely used in biosensing and other fields due to their ability to specifically recognize target molecules, often called "artificial receptors.". Nowadays, researchers are constantly exploring new design and synthesis methods for molecularly imprinted materials to improve the selectivity and sensitivity of molecularly imprinted materials. Among them, the selection of functional monomers has attracted great attention. This review comprehensively analyzes and discusses the selection methods of functional monomers. The most commonly used functional monomers among different types of templates are screened based on the structural properties of the template molecules, including the selection of functional monomers among ion-imprinted polymers, protein-imprinted polymers, and bacterial imprinted polymers. The rich binding sites and functional group types of multifunctional monomers are also highlighted to advance the development of molecular imprinting technology. The article further explores the current challenges and prospects in the selection of functional monomers and emphasizes multiplex experiments and computer simulations as important directions for future research. This review provides comprehensive information and constructive guidelines for researchers in selecting functional monomers in areas such as analytical chemistry and biosensors.

Recent Advances in Molecular Imprinting for Proteins on Magnetic Microspheres.

The separation of proteins in biological samples plays an essential role in the development of disease detection, drug discovery, and biological analysis. Protein imprinted polymers (PIPs) serve as a tool to capture target proteins specifically and selectively from complex media for separation purposes. Whereas conventional molecularly imprinted polymer is time-consuming in terms of incubation studies and solvent removal, magnetic particles are introduced using their magnetic properties for sedimentation and separation, resulting in saving extraction and centrifugation steps. Magnetic protein imprinted polymers (MPIPs), which combine molecularly imprinting materials with magnetic properties, have emerged as a new area of research hotspot. This review provides an overview of MPIPs for proteins, including synthesis, preparation strategies, and applications. Moreover, it also looks forward to the future directions for research in this emerging field.

Peptide-crosslinked IgG-imprinted polymers for antibody capture and separation

Downstream separation and purification of monoclonal antibodies (mAbs) has long been the major bottleneck for their production. Here novel molecularly imprinted polymers (MIPs) of immunoglobulin G (IgG) were designed for their capture and separation from cell culture supernatant. To overcome the severe problems faced by protein-imprinted polymers, i.e., difficult template removal and low imprinting efficiency, a poly(L-glutamic acid) (PLGA)-based peptide crosslinker, instead of commonly used crosslinkers, was employed. In addition, a thiophilic functional monomer, 2-((2-hydroxy-3-(methacryloyloxy)propyl)thio)nicotinic acid (GMA-NA), which can bind with IgG selectively, was designed and used for the synthesis of IgG-imprinted polymers. Because of the reversible and precise pH-induced helix-coil transition of the PLGA segments in the polymers and also the selective binding of GMA-NA with IgG, complete removal of the template protein was achieved under mild conditions. Meanwhile excellent imprinting efficiency was obtained. For the IgG-imprinted polymer with an optimized composition, a high adsorption capacity (790.2 mg/g) and a high IF (5.2) were achieved, both of which are much higher than the previously reported IgG-imprinted polymers. The polymer exhibits high selectivity towards the template IgG against non-template proteins. It also exhibits excellent reusability. The high adsorption capacity, high selectivity and mild elution conditions of the new IgG-imprinted polymer make it good adsorbent for antibody separation. Using the new MIP, IgG was successfully separated from human serum. More importantly, HLA-DRA mAb was successfully separated from cell culture supernatant with a removal rate of 89% and a purity of 87%, confirming the potential of the new MIP in mAb separation.

Molecularly Imprinted Polymers with Shape-Memorable Imprint Cavities for Efficient Separation of Hemoglobin from Blood.

Efficient separation and purification of hemoglobin from blood and other complicated biological fluids still remains a big challenge. Molecularly imprinted polymers (MIPs) of hemoglobin are potential choices; however, they suffer from severe problems including difficult template removal and low imprinting efficiency like other protein-imprinted polymers. Herein, a novel MIP of bovine hemoglobin (BHb) was designed in which a peptide crosslinker (PC), instead of the commonly used crosslinkers, was used. The PC, a random copolymer of lysine and alanine, adopts an α-helical conformation at pH 10 but transits to a random coil conformation at pH 5. The introduction of alanine residues lowers the pH range at which the PC undergoes helix-coil transition. The imprint cavities in the polymers are shape-memorable due to the reversible and precise helix-coil transition of the peptide segments in the polymers. They can be enlarged by lowering pH from 10 to 5, thus allowing complete removal of the template protein under mild conditions. When the pH is adjusted back to 10, their original size and shape will be recovered. Therefore, the MIP binds the template protein BHb with high affinity. Compared with the MIP crosslinked with the commonly used crosslinker, the imprinting efficiency of the PC-crosslinked MIP is significantly improved. In addition, both the maximum adsorption capacity (641.9 mg/g) and imprinting factor (7.2) are much higher than the BHb MIPs reported previously. The new BHb MIP also exhibits high selectivity toward BHb and good reusability. Thanks to the high adsorption capacity and high selectivity of the MIP, when it was applied to extract BHb from bovine blood, BHb in the blood sample was extracted almost completely, and high purity product was obtained.

Recent advances in protein-imprinted polymers: synthesis, applications and challenges.

The molecular imprinting technique (MIT), also described as the "lock to key" method, has been demonstrated as an effective tool for the creation of synthetic polymers with antibody-like sites to specifically recognize target molecules. To date, most successful molecular imprinting researches were limited to small molecules (<1500 Da); biomacromolecule (especially protein) imprinting remains a serious challenge due to their large size, chemical and structural complexity, and environmental instability. Nevertheless, protein imprinting has achieved some significant breakthroughs in imprinting methods and applications over the past decade. Some special protein-imprinted materials with outstanding properties have been developed and exhibited excellent potential in several advanced fields such as separation and purification, proteomics, biomarker detection, bioimaging and therapy. In this review, we critically and comprehensively surveyed the recent advances in protein imprinting, particularly emphasizing the significant progress in imprinting methods and highlighted applications. Finally, we summarize the major challenges remaining in protein imprinting and propose its development direction in the near future.

Construction of shape memorable imprinted cavities for protein recognition using oligo-l-lysine-based peptide crosslinker

Protein-imprinted polymers are artificial receptors capable of recognizing protein. They are highly promising for applications in important bio-related areas, however, their development was severely retarded by two problems: difficult template removal and low imprinting efficiency. The two problems could be overcome by constructing shape-memorable imprinted cavities using peptide crosslinker. Here a new oligo-l-lysine-based peptide crosslinker was designed and synthesized. A novel cytochrome c (Cyt C)-imprinted polymer was synthesized using the new peptide crosslinker. When switching pH between 12 and 7.4, the peptide segments incorporated in the polymer underwent reversible helix-coil transition. Because of the precise folding of the peptide segments, the imprinted cavities in the polymer could be enlarged when lowering pH to 7.4 to release the template protein, but restore their original size and shape at pH 12 to recognize the template protein. Therefore complete template removal was achieved under mild conditions. Meanwhile the imprinting efficiency was improved significantly. Compared to polymer crosslinked with the commonly used crosslinker N, N-methylenebisacrylamide, the imprinting efficiency of the peptide-crosslinked polymer was increased by 15 times. The new imprinted polymer presented not only a high adsorption capacity (454.4 mgg−1), a high imprinting factor (6.3), high selectivity towards Cyt C, and excellent reusability, but also could preserve the fragile secondary structure of the eluted protein, and therefore had high potential in bioseparation. As a demonstration, Cyt C added into fetal bovine serum was separated from the sample using the polymer via a simple adsorption-desorption cycle. The recovery rate was as high as 92.7%.

Peptide-Cross-Linked Protein-Imprinted Polymers: Easy Template Removal and Excellent Imprinting Effect

Molecular imprinting of proteins remains a huge challenge because of two major obstacles: difficulty in template removal and low imprinting efficiency. Herein, we propose a new strategy to simultan...

Molecularly imprinted polymer-based electrochemical sensors for biopolymers

Abstract Electrochemical synthesis and signal generation dominate among the almost 1200 articles published annually on protein-imprinted polymers. Such polymers can be easily prepared directly on the electrode surface, and the polymer thickness can be precisely adjusted to the size of the target to enable its free exchange. In this architecture, the molecularly imprinted polymer (MIP) layer represents only one ‘separation plate’; thus, the selectivity does not reach the values of ‘bulk’ measurements. The binding of target proteins can be detected straightforwardly by their modulating effect on the diffusional permeability of a redox marker through the thin MIP films. However, this generates an ‘overall apparent’ signal, which may include nonspecific interactions in the polymer layer and at the electrode surface. Certain targets, such as enzymes or redox active proteins, enables a more specific direct quantification of their binding to MIPs by in situ determination of the enzyme activity or direct electron transfer, respectively.

Correction to “Protein-Imprinted Polymers: The Shape of Things to Come?”

Correction to “Protein-Imprinted Polymers: The Shape of Things to Come?”

Coarse-Grained Simulation of Protein-Imprinted Hydrogels.

The MARTINI force-field is adapted for acrylic functional monomers and used to simulate protein-imprinted polymers, including complexation in solution, cross-linking reaction, washing, and rebinding of the protein. A globally controlled grand canonical Monte Carlo simulation combined with molecular dynamics is used to achieve proper hydration and swelling of the gel. Two gel formulations are compared, one with only polar functional monomers and one with additional small number of charged monomers. Imprinted hydrogels are evaluated for two proteins, lysozyme, and cytochrome c and chosen due to their similarity in size and isoelectric point, though it is known that their imprinting behavior is very different. Analysis of diffusion of the proteins within the gel shows that their interaction with functional monomers and their diffusion mechanism differ substantially and hence affect their specific and nonspecific interactions with the gel.

Magnetic protein imprinted polymers: a review.

Protein imprinted polymers have received a lot of interest in the past few years because of their applications as tailor-made receptors for biomacromolecules. Generally, the preparation of these polymers requires numerous and time-consuming steps. But their coupling with magnetic nanoparticles simplifies and speeds up the synthesis of these materials. Some recent papers describe the use of protein imprinted polymer (PIP) coupled to magnetic iron oxide nanoparticles (MION) for the design of MION@PIP biosensors. With such systems, a target protein can be specifically and selectively captured from complex media due to exceptional chemical properties of the polymer. Despite such performances, only a limited number of studies address these hybrid nanosystems. This review focuses on the chemistry and preparation of MION@PIP nanocomposites as well as on the metrics used to characterize their performances.

Selective binding of matrix metalloproteases MMP-9 and MMP-12 to inhibitor-assisted thermolysin-imprinted beads.

Protein-imprinted polymers have been synthesized to recognize and specifically bind selected proteins. However, protein imprinting requires substantial amounts of pure protein to efficiently obtain imprinted polymers for large scale applications, e.g. protein purification by affinity chromatography. In the absence of large quantities of a pure protein of interest, an alternative strategy was developed. In this case study, neutral metalloprotease thermolysin was selected as a commercially available surrogate for imprinting polymer beads. Phosphoramidon-assisted thermolysin-imprinted beads were synthesized. During rebinding experiments, it was shown that these beads specifically bind to thermolysin. In addition, it was shown that these beads also bind in CHO cell culture supernatant to the matrix metalloprotease-9 and -12 (MMP-9, -12). Therefore, these beads can be applied as a selective sorbent for the rare metalloproteases MMP-9 and MMP-12 to remove these proteases from CHO cell culture supernatants. The high selectivity of thermolysin-imprinted beads can be extended to other proteases of the family of metalloproteases, and is not limited to thermolysin. This innovative approach is suitable to address the challenges in the field of protease purification and isolation from biotechnologically relevant media.

Protein-Imprinted Polymers: The Shape of Things to Come?

The potential to develop materials with antibody-like molecular recognition properties has helped sustain interest in protein-imprinted polymers over the past several decades. Unfortunately, despite persistent research, the field of noncovalent protein imprinting has seen limited success in terms of achieving materials with high selectivity and high affinity. In this Perspective, important yet sometimes overlooked aspects of the imprinting and binding processes are reviewed to help understand why there has been limited success. In particular, the imprinting and binding processes are viewed through the scope of free radical polymerization and hydrogel swelling theories to underscore the complexity of the synthesis and behavior of protein-imprinted polymers. Additionally, we review the metrics of success commonly used in protein imprinting literature (i.e., adsorption capacity, imprinting factor, and selectivity factor) and consider the relevance of each to the characterization of an imprinted polymer's recognition characteristics. Throughout, common shortcomings are highlighted, and experiments that could help verify or disprove the efficacy of noncovalent protein imprinting are discussed.

Comparison of descriptors for predicting selectivity of protein-imprinted polymers.

Molecular imprinting is a technique that is used to create artificial receptors by the formation of a polymer network around a template molecule, creating a molecularly imprinted polymer. These artificial receptors may be used in applications that require molecular recognition, such as enantioseparations, biosensors, artificial catalysis, drug delivery and others. Small molecules, such as drugs, have been imprinted with high efficiency and, combined with the low cost of preparation, molecularly imprinted polymers have acquired commercial usage. While attempts at imprinting proteins have been significantly less successful, the great potential of protein-imprinted polymers (PIPs) in medicine and industry attracted much research. Multifunctionality, conformational flexibility, large size of the proteins, and aqueous polymerization environment are some of the obstacles faced by protein imprinting. We explore the relation between PIP selectivity and the properties of the template and competitor proteins. A comprehensive statistical analysis of published studies reveals a statistically significant correlation between four protein descriptors and the corresponding selectivity of PIPs. Namely, a PIP will generally be more selective against large competitor proteins with a smooth surface, whose isoelectric point and aspect ratio are significantly different than those of the template protein. The size of the protein, as measured by its molecular weight, appears to be independent of the template protein characteristics. Copyright © 2016 John Wiley & Sons, Ltd.

Protein imprinting over magnetic nanospheres via a surface grafted polymer for specific capture of hemoglobin

Combining surface molecular imprinting with a nano-sized physical form is an effective technology to achieve a large binding capacity and overcome template removal within molecularly imprinted polymers (MIPs), and is in particular advantageous to the template of bulky structures like proteins. In addition, the surface protein-imprinted polymers immobilized on nanospheres with magnetic function are more attractive in the research of selective recognition and enrichment of proteins. In this work, we have demonstrated a facile method, enhancing specific recognition of multifunctional molecularly imprinted magnetic nanospheres with core–shell structure. It was achieved simply via radical induced graft polymerization of low concentration monomers on the surface of vinyl groups modified magnetic nanospheres dispersed in aqueous media using hemoglobin (Hb) as a template. The possible gelation of the dispersion after polymerization was avoided and hence the imprinted magnetic particles without an obvious agglomerated phenomenon could be collected readily and expediently. Finally, a highly monodisperse magnetic nanospheres were synthesized successfully. The morphological studies of the resultant MIPs-coated magnetic particles were characterized using various methods. In batch rebinding tests, the imprinted magnetic particles reached saturated adsorption within 30 min and exhibited significant specific recognition towards the target protein molecules with a binding capacity as high as 281.94 mg g−1 and an imprinting factor of 4.29. The resultant Hb-MIPs magnetic particles not only extracted target protein from mixed proteins, but also enriched the low abundance of hemoglobin. Moreover, the stability and regeneration were also investigated, which indicated the excellent property of reutilization.

Fluorescent protein-imprinted polymers capable of signal transduction of specific binding events prepared by a site-directed two-step post-imprinting modification

Protein recognition polymers capable of highly specific transduction of protein binding events into fluorescence change were prepared by molecular imprinting in conjunction with a newly developed two-step post-imprinting chemical modification of functional groups located within the protein recognition cavity.

Preparation and application of hollow molecularly imprinted polymers with a super‐high selectivity to the template protein

Protein-imprinted polymers with hollow cores that have a super-high imprinting factor were prepared by etching the core of the surface-imprinted polymers that used silica particles as the support. Lysozyme as template was modified onto the surface of silica particles by a covalent method, and after polymerization and the removal of template molecules, channels through the polymer layer were formed, which allowed a single-protein molecule to come into the hollow core and attach to the binding sites inside the polymer layer. The adsorption experiments demonstrated that the hollow imprinted polymers had an extremely high binding capacity and selectivity, and thus a super-high imprinting factor was obtained. The as-prepared imprinted polymers were used to separate the template lysozyme from egg white successfully, indicating its high selectivity and potential application in the field of separation of protein from real samples.

Sample-Imprinted Polymer Potentially for Protein Depletion and Enrichment

Here we report a method to prepare protein-imprinted polymers in the presence of a real sample (chicken egg white), rather than any known purified proteins. By comparing the electrophoretic separation of the sample in the imprinted polymer and a control capillary, it's found such a sample-imprinted polymer could retain abundant species. As a result the signals of abundant proteins in the sample were removed or reduced in the electropherogram. Meanwhile new peaks appeared to stand for enrichment of the minor proteins in the same sample. This suggests a new way to investigate molecular imprinting, which means analyzing complicated samples based on their instinctive complexities, i.e., a series of polymers just imprinted by themselves.

Pending templates imprinted polymers—hypothesis, synthesis, adsorption, and chromatographic properties

This is the first time when protein-imprinted polymers are prepared with "pending templates." The polymers were synthesized in the presence of a real sample (chicken egg white), rather than any known commercial proteins. Compared with a simultaneously synthesized nonimprinted control polymer, the polymers show higher adsorption capacity for abundant components (as "pending templates") in the original sample. Chromatography experiments indicated that the columns made of the imprinted polymers could retain abundant species (imprinted) and separate them from those not imprinted. Thus, the sample could be split into dimidiate subfractions with reduced complexities. "Pending template imprinting" suggests a new way to investigate molecular imprinting, especially to dissect, simplify, and analyze complicated samples through a series of polymers just imprinted by the samples per se.

Fluorescent molecularly imprinted polymer thin films for specific protein detection prepared with dansyl ethylenediamine-conjugated O-acryloyl l-hydroxyproline

Protein-imprinted polymers, capable of specific transduction of protein binding events into fluorescent signal change, were designed and synthesized by using dansyl ethylenediamine-conjugated O-acryloyl l-hydroxyproline (Hyp-En-Dans). Human serum albumin (HSA) was used as a model target protein and HSA-imprinted polymers (HSA-IP) were prepared on glass substrates. Specific fluorescence change was observed for HSA binding on the imprinted polymer thin film, whereas a weaker response was observed for other proteins, including bovine serum albumin, chymotrypsin, lysozyme, and avidin. The binding specificity was found to derive from the rigid structure of the hydrogen-bondable pyrrolidine moiety. Compared with SPR measurements, the non-specific binding caused by the polymer matrix and/or randomly located fluorescent monomer residues that did not compose specific binding sites did not contribute to the observed fluorescence change. These results revealed that the proposed protein-imprinting technique using Hyp-En-Dans could provide a highly selective protein-sensing platform, in which only specific binding events would be detected by fluorescent measurements.