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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.

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The development of high-performance protein-imprinted materials remains challenging due to defects concerning high mass transfer resistance and non-specific binding, which are crucial for protein purification and enrichment. In this paper, lysozyme-imprinted mesoporous Zr-based MOF (mesoUiO-66-NH2@MIPs) with specific and selective recognition of lysozyme (Lyz) were prepared by surface imprinting technology. In particular, the excellent hydrophilicity mesoporous MOFs (mesoUiO-66-NH2) with a pore size of 10 nm was prepared as a carrier for Lyz immobilization by an auxiliary modulation strategy to regulate the microporous structure of UiO-66-NH2 with the propionic acid solution, enabling massive loading of the macromolecular protein Lyz. The mesoUiO-66-NH2@MIPs reached a maximum saturation adsorption of 206.54 mg g-1 on Lyz in 20 min at 25 °C with an imprinting factor of 2.57 and selection factors of 2.02, 2.34, and 2.45 for cytochrome c (Cyt c), bovine serum albumin (BSA) and bovine hemoglobin (BHb), respectively. More importantly, the mesoUiO-66-NH2@MIPs could specifically recognize Lyz from the mixed protein system. The adsorption capacity of Lyz could still reach 78.55% after 5 cycles with good cyclic regeneration performance. This provides a new research option for developing and applying novel porous MOF in biomolecule imprinting technology and the specific separation of biomolecules.

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In this study, protein-imprinted sensors were electrochemically fabricated on screen-printed carbon electrodes (SPCEs) for the cytokine interleukin-1ß (IL-1ß) detection. A double layer comprising poly(o-phenylenediamine) and poly(chromotrope 2R) with a template (i.e., IL-1ß biomacromolecules) was formed through the cyclic voltammetry (CV) technique to modify the molecularly imprinted polymer (MIP) films on the SPCEs. The electrochemical sensing properties were investigated via CV and electrochemical impedance spectroscopy to confirm the imprinting effect on the MIP films. The results show that the MIP sensor has a highly sensitive response in the trace IL-1ß solution (a few pg/mL) with a limit of detection of 0.23 pg/mL and a limit of quantification of 0.78 pg/mL. Furthermore, the MIP sensor showed high selectivity for IL-1ß adsorption compared to other proteins. In summary, based on binary double layers, the impedance sensing platforms of electropolymerized MIP films show potential application in the practical detection of macromolecular proteins.

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In conjunction with polyacrylamide gel electrophoresis (PAGE), molecular imprinting methods have been applied to produce a multilayer mini-slab in order to evaluate how selectively and specifically a hydrogel-based molecularly imprinted polymer (MIP) binds bovine haemoglobin (BHb, ~64.5 kDa). A three-layer mini-slab comprising an upper and lower layer and a MIP, or a non-imprinted control polymer dispersion middle layer has been investigated. The discriminating MIP layer, also based on polyacrylamide, was able to specifically bind BHb molecules in preference to a protein similar in molecular weight such as bovine serum albumin (BSA, ~66 kDa). Protein staining allowed us to visualise the protein retention strength of the MIP layer under the influence of an electric field. This method could be applied to other proteins with implications in effective protein capture, disease diagnostics, and protein analysis.

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Molecularly imprinted polymers endowed with photo-luminescent properties have attracted wide research interest in many fields such as biological analysis and diseases diagnosis. Herein, we illustrate a versatile method for the construction of surface protein-imprinted nanoparticles based on metal coordination and anchored carbon dots (CDs) for enhanced fluorescence detection of the target protein. As the fluorescent nanosupports for surface imprinting, CDs-attached SiO2 nanoparticles were synthesized via thiol-ene click chemistry. With histidine (His)-exposed protein as templates, imprinted nanoshells were formed over the nanosupports via copolymerization of a Cu2+-chelating monomer and an oligo (ethylene glycol) monomer, hence producing high-quality imprinted cavities because of both the relatively strong coordination and inhibited non-specific binding. Using lysozyme as a model His-exposed template, the imprinted nanoparticles showed fluorescence enhancement while binding the target protein, and exhibited significantly increased specific fluorescence response than the controls without the metal coordination. They achieved a high imprinting factor of 5.8 and a low limit of detection of 10.1 nM. Furthermore, such sensors were applied to determine lysozyme in diluted chicken egg-white samples with satisfactory recoveries at three spiking levels ranging from 97.9 to 101.4%. Human serum albumin was also used as another template protein for preliminary confirming the generality of the presented strategy.

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Protein imprinted MIPs show notable potential for applications in many analytical areas such as clinical analysis, medical diagnostics and environmental monitoring, but also in drug delivery scenarios. In this study, we present various modifications of two different synthesis routes to create imprinted core-shell particles serving as a synthetic recognition material for the protein hen egg white (HEW) lysozyme. HEW lysozyme is used as food additive E 1105 for preservation due to its antibacterial effects. For facilitating quality and regulatory control analysis in food matrices, it is necessary to apply suitable isolation methods as potentially provided by molecularly imprinted materials. The highest binding capacity achieved herein was 58.82 mg/g with imprinting factors ranging up to 2.74, rendering these materials exceptionally suitable for selectively isolating HEW lysozyme.

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Epitope imprinting is an effective strategy to prepare molecularly imprinted polymers (MIPs) for protein recognition. Indeed, the idea to use as a template just a fragment of the protein of interest, called the epitope, instead of the whole protein, presents some key advantages for the imprinting process, in particular: cutting the costs for MIP production and avoiding protein unfolding during the imprinting process, so to ultimately improve the quality of the stamped binding sites. How to select an epitope for the imprinting is the strategic question. Here, the bioinformatics tools to search for suitable epitopes for the imprinting process and rational tools to select the most suitable epitope are briefly introduced along with protocols for their practical use.

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To develop multifunctional protein imprinted materials, a cobalt-iron double ion-BSA directional chelation-assisted thermo-sensitive surface-imprinted hollow nanocage (Co-Fe@CBMA-MIPs) with excellent specificity is developed on the surface of ZIF-67@Co-Fe in this study by synergizing the advantages of surface imprinting, metal ion chelation, anti-protein adsorption segments, and thermo-sensitive components. Beyond previous research, well-designed multifunctional protein-imprinted materials possess high binding capacity, fast adsorption kinetics, and outstanding selectivity. When the adsorption is carried out at 32 °C, the adsorption capacity of Co-Fe@CBMA-MIPs for BSA reaches 520.35 mg/g within 50 min. The imprinting factor is 8.55. The selectivity factors of Co-Fe@CBMA-MIPs for HSA, Bhb, OVA, and Lyz are 3.72, 6.09, 4.10, and 8.41, respectively. More significantly, Co-Fe@CBMA-MIPs could specifically recognize BSA from mixed proteins and actual samples and exhibit excellent repeated use stability. Based on the above advantages, the development of this research provides an effective means to improve the recognition specificity of molecularly imprinted polymers.

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The molecular imprinting of proteins is the process of forming biomimetics with entailed protein-recognition by means of a template-assisted synthesis. Protein-imprinted polymers (pMIPs) have been successfully employed in separations, assays, sensors, and imaging. From a technical point of view, imprinting a protein is both costly, for protein expression and purification, and challenging, for the preservation of the protein's structural properties. In fact, the imprinting process needs to guarantee the preservation of the same protein three-dimensional conformation that later would be recognized. So far, the captivating idea to imprint just a portion of the protein, i.e., an epitope, instead of the whole, proved successful, offering reduced costs, compatibility with many synthetic conditions (solvents, pH, temperatures), and fine-tuning of the peptide sequence so to target specific physiological and functional conditions of the protein, such as post-translational modifications. Here, protein-protein interactions and the biochemical features of the epitopes are inspected, deriving lessons to prepare more effective pMIPs. Epitopes are categorized in linear or structured, immunogenic or not, located at the protein's surface or buried in its core and the imprinting strategies are discussed. Moreover, attention is given to freely available online bioinformatics resources that might offer key tools to gain further rationale amid the selection process of suitable epitopes templates.

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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%.

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In this study, a selective and sensitive molecular imprinting-based electrochemical sensors, for horseradish peroxidase (HRP) entrapment was fabricated using electro polymerization of ß-Cyclodextrin (ß-CD) on the surface of glassy carbon electrode. Poly beta-cyclodextrin P(ß-CD) provide efficient surface area for self-immobilization of HRP as well as improve imprinting efficiency. The proposed imprinted biosensor successfully utilized for detection of HRP with excellent analytical results which linear range is 0.1 mg/mL to 10 ng/mL with LOD of 2.23 ng/mL. Furthermore, electrocatalytical activity of the prepared biosensor toward the reduction of hydrogen peroxide was investigated in the ranges of 1 to 15 µM with a detection limit of 0.4 µM by using chronoamperometry technique. The developed biosensor was used for the detection of hydrogen peroxide in unprocessed human plasma sample.

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Molecularly imprinted polymer nanoparticles (nanoMIPs) are receiving broad interest as robust and highly selective synthetic receptors for a variety of molecules. Due to their stability, inexpensive synthesis and easy implementation, they are considered a promising alternative to antibodies in sensors, diagnostics and separation applications. The most challenging targets for the production of synthetic receptors are proteins due to their fragile nature and the multitude of possible binding sites in their structure. Herein, we describe the modification and optimization of the protocol for synthesis of nanoMIPs with specificity for proteins using the prototype of an automated solid-phase synthesizer. Using an automated system gives an advantage for the simple, fast and fully controlled, reproducible production of nanoMIPs. The molecular imprinting in the reactor is performed using a template covalently immobilized on a solid support, in mild conditions suitable for preserving protein native structure. The validation of the protocol was made by assessing the ability to regenerate a solid-phase, and by measuring affinity and specificity of nanoparticles. As a model protein, we have chosen trypsin since its enzymatic activity can be easily monitored by using a commercial colorimetric assay. Different protocols were tested for their ability to improve the yield of high affinity nanoparticles in the final elution.

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To promote the development of molecular imprinting technique in the separation and analysis of protein, novel bovine serum albumin (BSA) surface imprinted nitrogen-doped magnetic carbon nanotubes (N-MCNTs@MIPs) are developed by this paper. The imprinted materials are prepared by depositing polydopamine (PDA) on the surface of nitrogen-doped magnetic carbon nanotubes (N-MCNTs). N-MCNTs prepared by high temperature pyrolysis and chemical vapor deposition exhibit high specific surface area, positive hydrophilicity, abundant nitrogen functional groups and excellent magnetic properties. These characteristics are conducive to the increase of effective binding sites, the smooth development of the protein imprinting process in the aqueous phase, the improvement of the binding capacity and the simplification of the separation process. The amount of BSA adsorbing on the N-MCNTs@MIPs can reach 150.86 mg/g within 90 min. The imprinting factor (IF) is 1.43. The results of competitive adsorption and separation of fetal bovine serum showed that N-MCNTs@MIPs can specifically recognize BSA. The excellent reusability and separation ability for real sample prove that N-MCNTs@MIPs have the potential to be applied to the separation and purification of proteins in complex biological samples.

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Molecularly imprinted polymer nanoparticles (MIP NPs) are antibody-like recognition materials prepared by a template-assisted synthesis. MIP NPs able to target biomolecules, like proteins, are under the spotlight for their great potential in medicine, but efficiently imprinting biological templates is still very challenging. Here we propose generating a molecular imprint in single NPs, by photochemically initiating the polymerization from individual protein templates. In this way, each protein molecule tailors itself its own "polymeric dress". For this, the template protein is covalently coupled with a photoinitiator, Eosin Y. Irradiated with light at 533 nm, the Eosin moiety acts as an antenna and transfers energy to a co-initiator (an amine), which generates a radical and initiates polymerization. As a result, a polymer network is forming only around the very template molecule, producing cross-linked NPs of 50 nm, with single binding sites showing high affinity (KD 10-9 m) for their biological target, and selectivity over other proteins.

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Constructing imprinting materials with high recognition and selectivity for protein is an always challenge in protein imprinting technology (PIT). In this work, upon the participating of a zwitterionic polymer chain (Poly (1-vinyl-3-sulfopropylimidazolium), PVSP), a lysozyme imprinted core-shell carbon microsphere (CFC-PVSP@MIPs) was prepared by combining template immobilization method and surface imprinting technology. The carboxyl-functionalized carbon microspheres as substrate provided the CFC-PVSP@MIPs satisfactory adsorption capacity (68.1 mg g-1), while the dopamine as a functional monomer and crosslinker allowed the imprinted microspheres to have a thin imprinted shell, thus endowing them a fast adsorption equilibrium rate (120 min). In addition, PVSP could be tightly bound to the imprinted layer through non-covalent interaction, which not only simplified the preparation process of CFC-PVSP@MIPs, but also reduced the non-specific adsorption of imprinted material on proteins. Therefore, the resulting CFC-PVSP@MIPs exhibited a more superior recognition ability towards lysozyme with imprinting factor value of 3.10, compared with the PVSP-free imprinted microsphere (imprinting factor value 1.93). Furthermore, benefiting from the characteristics of zwitterionic groups, CFC-PVSP@MIPs also revealed stronger selectivity in competitive adsorption studies of binary protein mixture samples. Consequently, the proposed strategy would be a promising and convenient way to obtain protein imprinted material with high recognition ability, thus would be conducive to further development and application of PIT.

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Along with the wide development of protein imprinted polymers, the researchers still face many challenges, such as difficult template elution, slow adsorption rate and low adsorption capacity. In order to promote the progress of protein separation and purification, the surface imprinted manganese dioxide-loaded tubular carbon fibers (FTCFs@MnO2@MIPs) are prepared in this work. FTCFs@MnO2@MIPs are based on tubular carbon fibers (TCFs) coated with flaky MnO2. Dopamine (DA) and bovine serum albumin (BSA) are utilized as functional monomers and templates. The MnO2 nanosheets are grown and loaded on the surface of carboxyl-modified tubular carbon fibers (CMTCFs) to form a MnO2 shell, which provides more imprinting sites for protein imprinting. Meanwhile, this shell enhances the interaction between the imprinting sites and BSA. The content of MnO2 loaded on the surface of CMTCFs is 9.42%. The obtained materials are systematically characterized and the adsorption performances of FTCFs@MnO2@MIPs for BSA are investigated. The adsorption process of FTCFs@MnO2@MIPs exhibits significant self-driven characteristics. The adsorption capacity reaches 816.44 mg/g in 60 min and the imprinting factor (IF) is 3.31. FTCFs@MnO2@MIPs can selectively separate BSA from the mixed proteins and fetal bovine serum. Excellent reusability and practical application ability make MnO2-loaded tubular carbon fibers (FTCFs@MnO2) become a promising carrier in the field of protein imprinting.

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One viable solution to improve the conformational stability of template proteins is to use multiple, weaker modes of action to immobilize proteins on the surfaces of a solid support. Herein, we introduce a novel surface imprinting technique for human serum albumin (HSA) by a dual immobilization/imprinting strategy. Specifically, HSA was first conjugated to the surfaces of magnetic Fe3O4 nanoparticles through a reversible aldmine condensation reaction. Dopamine (DA) was then used to imprint the protein template via an auto-polymerization reaction in biocompatible aqueous media. The resultant magnetic molecular imprinted polymers (MMIPs) possess high adsorption capacity (70.2 mg g-1), superior selectivity (IF = 4.54), and rapid capturing kinetics to HSA (within 20 min). We successfully demonstrate the practical applicability of MMIPs to the selective removal of HSA from human serum sample. Our work offers a novel and robust solution to develop proteins imprinted materials with high binding capacity and selectivity. We anticipate such materials will find wide applications to protein detection or removal in diverse real-life clinical and biological samples.

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Materials that can mimic the molecular recognition-based functions found in biology are a significant goal for science and technology. Molecular imprinting is a technology that addresses this challenge by providing polymeric materials with antibody-like recognition characteristics. Recently, significant progress has been achieved in solving many of the practical problems traditionally associated with molecularly imprinted polymers (MIPs), such as difficulties with imprinting of proteins, poor compatibility with aqueous environments, template leakage, and the presence of heterogeneous populations of binding sites in the polymers that contribute to high levels of non-specific binding. This success is closely related to the technology-driven shift in MIP research from traditional bulk polymer formats into the nanomaterial domain. The aim of this article is to throw light on recent developments in this field and to present a critical discussion of the current state of molecular imprinting and its potential in real world applications.

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In this work, a novel thermosensitive surface protein imprinted polymer monolithic column (TsIPMC) was synthesized by combining high internal phase emulsion with 1,1-diphenylethene (DPE) controlled polymerization. Innovatively, DPE and acrylic acid (AA) monomers were introduced in high internal oil and water phases respectively. The research showed that DPE could not only initiate the polymerization of monomers, but also improve the pore performance of monolithic columns. The elution efficiency of template or target protein could be significantly improved by the thermoresponse characteristics of TsIPMC. The effects of DPE and AA on adsorption capacity and imprinting factor (IF) were studied. The optimization results presented that the optimal addition amounts were 55 mg and 50 mg. Under such conditions, the IF of as-prepared TsIPMC was 1.61 and the saturated adsorption capacity was 66 mg/mL. The influences of the flow rate and target protein concentration on the adsorption equilibrium time and effluent volume were revealed. TsIPMC showed higher selectivity for different competing proteins. The reuse stability result showed that the adsorption of TsIPMC to BSA decreased by 3.69% after 12 times of reuse, and the IF remained basically unchanged. TsIPMC would demonstrate the potential applications in the field of protein purification and separation.

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An imprinting technique has been developed to generate synthetic polymer beads suitable for selectively binding a supramolecular target. The viral hexon protein, which is the most abundant and accessible surface protein component of the human Adenovirus type 5 (hAdV5) icosahedral capsid, was applied as the template molecule to generate functional polymer beads entailing selectivity for the entire virus. Individual and competitive rebinding studies using two different viruses (i.e. hAdV5 and Minute Virus of Mice - MVM) revealed exquisite selectivity of the imprinted beads for the target hAdV5. Additionally, the morphology of thus imprinted beads was checked via scanning electron microscopy (SEM).

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