Recent Advances in Macromolecularly Imprinted Polymers by Controlled Radical Polymerization Techniques

Mol. Impr. 2015; 3: 35–46

Review Article Open Access

Huiqi Zhang* Recent Advances in Macromolecularly Imprinted Polymers by Controlled Radical Polymerization Techniques

DOI 10.1515/molim-2015-0005 success in the practical applications of such biological Received August 11, 2015; accepted Ocotber 27, 2015 receptors, their inherent drawbacks, including poor physical Abstract: Molecularly imprinted polymers (MIPs) are and chemical stability, batch-to-batch variation and high synthetic receptors with tailor-made recognition sites for cost, significantly limit their more broad uses. To address the target molecules. Their high molecular recognition this issue, many efforts have been devoted to developing ability, good stability, easy preparation, and low cost strategies for the preparation of synthetic receptors with make them highly promising substitutes for biological an affinity and specificity approaching those achieved in receptors. Recent years have witnessed rapidly increasing nature. One strategy that has attracted rapidly increasing interest in the imprinting of biomacromolecules and interest nowadays is the molecular imprinting technique, especially proteins because of the great potential of which has been demonstrated to be highly versatile for these MIPs in such applications as proteome analysis, preparing synthetic receptors (or namely molecularly clinical diagnostics, and biomedicine. So far, some imprinted polymers (MIPs)) with tailor-made recognition useful strategies have been developed for the imprinting sites for the target molecules [5-15]. The resulting MIPs of proteins and controlled radical polymerization have proven promising substitutes for biological receptors techniques have proven highly versatile for such purpose. because of their high specific molecular recognition ability, This mini-review describes recent developments in the good stability, ease of preparation, and low cost, and they controlled preparation of proteins-imprinted polymers have shown great potential in such areas as separation via such advanced polymerization techniques. and purification, antibody mimics (immunoassay or biomedicine), chemical sensors, biomimetic catalysis, drug Keywords: Molecularly imprinted polymers, Synthetic development, and drug delivery. receptors, Proteins, Controlled radical polymerization The molecular imprinting technique can be simply techniques. defined as a template-induced polymerization approach for the generation of synthetic receptors. It typically involves the copolymerization of a functional monomer and a crosslinking monomer in the presence of a target 1 Introduction analyte called a “template” in a porogenic solvent, and the subsequent removal of the template from the resulting In nature, biological receptors with high specific molecular crosslinked polymer networks leads to MIPs with binding recognition ability (e.g. antibody and enzyme) play an sites complementary to the shape, size and functionality important role in virtually all life processes [1]. They have of the template (Fig. 1). So far, three types of molecular drawn great attention in both research and industry because imprinting approaches (covalent, noncovalent, and semi- of their high impact in selective separations, catalytic covalent) have been developed on the basis of the different processes, and sensitive chemical assays [2-4]. Despite much interactions used between the template and functional monomer (or functional group in the binding sites) during *Corresponding author Huiqi Zhang, Key Laboratory of Functional the imprinting and rebinding steps [8,9]. Among them, Polymer Materials (Ministry of Education), State Key Laboratory the noncovalent molecular imprinting approach is mostly of Medicinal Chemical Biology, Collaborative Innovation Center used nowadays because it can be easily accomplished of Chemical Science and Engineering (Tianjin), and College of without the need of complicated organic syntheses and is Chemistry, Nankai University, Tianjin 300071, P. R. China. E-mail: [email protected]. applicable to a wide range of template molecules.

Figure 1. Schematic representation for the generation of molecularly imprinted polymers: (a) complex formation; (b) copolymerization; (c) removal of template and (d) template rebinding. Adapted with permission of Elsevier B. V. from [14].

So far, a great number of MIPs have been designed rather hard to control with regard to chain propagation and for many different template molecules including both termination, which makes it difficult to prepare surface- small analytes (molecular weight (MW) < 1500 Da, such as imprinted polymer layers with well-defined and desired amino acids, drugs, pesticides, etc.), biomacromolecules thickness. In addition, they normally lead to crosslinked (MW > 1500 Da, such as proteins and polypeptides), and polymer networks with heterogeneous structures [23], even living organisms (e.g. bacteria). Despite significant which might be responsible for some of the inherent progress made in the molecular imprinting of small drawbacks of the MIPs such as the broad binding site templates and the successful commercial application of heterogeneity and the relatively low affinity and selectivity. MIPs in solid-phase extraction area in the past four decades, Therefore, it can be envisioned that the controlled the imprinting of biomacromolecules remains a big preparation of well-defined MIPs with homogeneous challenge, mainly because of their large sizes, complexed network structures will be of significant importance both for structures, and conformational variability [16-21]. better understanding the structure-property relationship of These inherent characteristics of biomacromolecules the MIPs and for obtaining MIPs with improved binding lead to many significant problems for the resulting properties. In this respect, CRPs are perfectly suited for macromolecularly imprinted polymers (mMIPs) such as the this purpose. The negligible chain termination in CRPs laborious removal of large templates and their slow access and their thermodynamically controlled processes allow to the binding sites as well as the difficult choice of suitable a more constant rate for the polymer chain growth, functional monomers and polymerization conditions (for leading to homogeneous polymer networks with a narrow example several functional monomers are usually used distribution of the network chain length. Many different together for the imprinting of proteins instead of using polymer networks with homogeneous structures have been only one as in conventional molecular imprinting, and prepared via CRPs [23-26]. careful choice of pH and salt concentration is necessary for CRPs have drawn great attention over the past decades preserving the native conformation of the template protein for providing simple and robust routes to the synthesis of and for controlling the charge state of the protein). well-defined polymers [27-31]. They have now become one The past three decades have witnessed considerable of the most rapidly developing areas in the field of polymer efforts being devoted to addressing the above-mentioned science. In comparison with living anionic polymerization, problems. Some promising strategies (including the surface CRPs can now offer similar control over the synthetic imprinting and epitope imprinting approaches [9,16-21]) process, although with somehow lower precision. On the have been developed for the synthesis of mMIPs to solve the other hand, CRPs can offer great advantages in terms of problems caused by the large sizes of biomacromolecules their much milder and less restricted reaction conditions (i.e. difficult removal of such large templates and their and their applicability to a larger range of monomers. In slow access to the binding sites). In addition, recent analogue with living cationic polymerization, the control years have also seen rapidly increasing interest in the over CRPs depends on creating a dynamic equilibrium application of controlled/“living” radical polymerization between the active and dormant species (Scheme 1a-d), techniques (CRPs) in the imprinting of biomacromolecules which eventually leads to negligible radical termination and especially proteins [22]. It is well known that MIPs and thus controlled polymerizations. Nowadays, the are normally prepared by a conventional free radical most investigated CRPs include iniferter-induced radical polymerization mechanism, mainly due to its tolerance of polymerization [27], atom transfer radical polymerization a wide range of functional groups in the monomers and (ATRP) [28,29], reversible addition-fragmentation chain templates as well as its mild reaction conditions. However, transfer (RAFT) polymerization [30], and nitroxide- conventional radical polymerization processes are usually mediated living radical polymerization (NMP) [31]. So Recent Advances in Macromolecularly Imprinted Polymers by Controlled Radical Polymerization Techniques 37

Addition * Fragmentation c) Pn* + S C S R Pn S C S R Pn S C S + R* Addition Z Fragmentation Z Z

Propagating CTA Intermediate Dormant Fragment radical radical species radical

Scheme 1. The mechanism of controlled radical polymerization techniques including (a) iniferter-induced radical polymerization, (b) ATRP, Scheme(c) RAFT polymerization,1. The mechanism and (d) NMP. of controlled radical polymerization techniques including (a) iniferter-induced radical polymerization, (b) ATRP, (c) RAFT polymerization, and (d) NMP. far, all these CRPs have been applied in the molecular TERminator in the process of radical polymerization. imprinting field (mostly for small templates), leading to Under suitable conditions, an iniferter decomposes MIPs with controlled structures and improved binding into an active radical and an inactive radical, where the properties in most cases. In the following section, we active radical initiates the polymerization of monomers present a summary of the recent advances in the controlled and the inactive radical functions as the capping agent preparation of mMIPs (or protein-imprinted polymers) via for propagating radicals to form dormant species. The CRPs (note that no report on the preparation of mMIPs via presence of the dynamic equilibrium between the dormant NMP has been disclosed yet). species (iniferters) and active species (propagating radicals) is responsible for the controllability of iniferter- induced radical polymerization (Scheme 1a). Similar 2 Application of CRPs in the prepa- to normal radical initiators, there are also thermal or ration of mMIPs or protein-imprin- photoiniferters. Among the various iniferters developed, photoiniferters have proven to be more efficient in ted polymers inducing controlled polymerizations in comparison to thermal ones and the well-designed dithiocarbamates 2.1 Application of iniferter-induced radical are the most efficient photoiniferters, inducing controlled polymerization radical polymerization that leads to various functional, block, graft, star, and crosslinked polymers, though with Iniferter-induced radical polymerization was developed relatively broad molecular weight distributions. by Ostu and coworkers in 1982 [27], where an iniferter24 Since the first application of iniferter-induced can concurrently act as the INItiator, transFER agent and radical polymerization in the molecular imprinting 38 H. Zhang field [32], many MIPs have been synthesized using this monomer, N,N’-methylene bisacrylamide (MBA) as the approach, where the templates are mostly small organic crosslinker, and phosphate buffer solution (PBS, pH 5.4) ones [22]. Only a few protein-imprinted polymers (or as the solvent. The resulting BHb-MIP particles proved protein-MIPs) have been prepared via iniferter-induced to show relatively fast binding kinetics, high template radical polymerization up to now [33-37]. He, Li and binding capacities and good selectivity. Similarly, Zhang’s group reported successful synthesis of the lysozyme-MIP microspheres were also prepared with MCP first series of protein-MIPs via iniferter-induced radical beads as the supporting particles and their applicability in polymerization following the route shown in Fig. 2 [33,34]. chromatographic separation of the template protein from In one of their works [33], mesoporous chloromethylated some competitive proteins (including bovine hemoglobin, polystyrene (MCP) beads were first modified with bovine serum albumin, ovalbumin or cytochrome c) in diethyldithiocarbamate sodium to introduce aqueous mobile phase was confirmed [34]. Moreover, dithiocarbamate groups on their surfaces, and they were lysozyme-MIP microspheres with double thermosensitive subsequently utilized as the immobilized iniferter to gates were also synthesized by the same group [35]. The induce surface protein imprinting at room temperature MCP beads with surface-bound dithiocarbamate groups under UV light irradiation with bovine hemoglobin (BHb) were again used as the immobilized iniferter to initiate as the template, acrylamide (AAm) as the functional the surface-imprinting of lysozyme with a mixture of

Figure 2. Schematic representation for the preparation of protein-MIP microspheres by surface iniferter-induced radical polymerization. Reproduced with permission of Elsevier B. V. from [34]. Recent Advances in Macromolecularly Imprinted Polymers by Controlled Radical Polymerization Techniques 39

N-isopropylacrylamide (NIPAAm), AAm, and methacrylic 2.2 Application of ATRP acid (MAA) as the co-functional monomers, MBA as the crosslinker, and tris(hydroxymethyl)aminomethane- Since its first discovery in 1995, ATRP has attracted hydrochloric acid (Tris-HCl) buffer (10 mM, pH 7.0) as the great attention over the past two decades due to its solvent. The resulting lysozyme-MIP microspheres were versatility in preparing well-defined polymers under mild further grafted with poly(NIPAAm) through the second- reaction conditions [28,29,38,39]. It is based on a fast, step photoinduced iniferter radical polymerization dynamic equilibrium established between the dormant of NIPAAm. Improved lysozyme selectivity and species (e.g. alkyl halides) and active species (radicals), thermosensitive properties were observed for such with transition-metal complexes acting as reversible protein-MIP particles, and their temperature-controlled halogen atom transfer reagents (Scheme 1b), which release of the template protein and reference proteins was keeps a very low radical concentration in the system also demonstrated. Furthermore, Li and coworkers also and thus results in negligible radical termination and described the fabrication of lysozyme-MIP membranes controlled polymerization. The feasibility of performing for the selective transport and separation of lysozyme ATRP under mild reaction conditions (e.g. at room by the first generation of a poly(acrylonitrile-co-N,N’- temperature and in aqueous media) and its applicability diethylaminodithiocarbamoylmethylstyrene) [P(AN-co- to a wide range of functional monomers [28,29,38,39] and DTCS)] membrane via the phase inversion method and biomacromolecules [40-43] make it a highly promising the subsequent grafting of a lysozyme-MIP layer on it by technique for molecular imprinting and in particular for surface-initiated iniferter radical polymerization (with imprinting of proteins. AAm as the functional monomer, MBA as the crosslinker, Husson and coworkers reported the first successful and PBS (10 mM, pH 6.2) as the solvent) [36]. A selectivity application of ATRP in the molecular imprinting field in factor of 2.51 and 2.13 was obtained for the lysozyme-MIP 2005 [44]. They prepared ultrathin (< 10 nm) MIP films membrane during the separation of a mixture of lysozyme on the surfaces of gold-coated silica wafers via surface- and bovine hemoglobin and a mixture of lysozyme and initiated ATRP with some fluorescently labeled amino acids cytochrome c, respectively. as the templates. Following this pioneering work, many Very recently, Madhuri and coworkers described the advanced MIPs have been prepared via ATRP for a wide construction of a sensitive and selective electrochemical range of template molecules including both small organic sensor for the detection of prostate specific antigen ones and proteins [22]. So far, two main strategies have (PSA) by the combined use of iniferter-induced radical been developed to prepare protein-imprinted polymers polymerization, surface imprinting techniques and via ATRP [45-53]. In the first strategy, protein-MIPs were nanotechnology strategies [37]. The multiwalled carbon prepared through two steps by using the scaffold imprinting nanotubes (MWCNTs) were first functionalized with concept. It is known that some functional monomers dithiocarbamate groups and then decorated with (e.g., MAA, AAm, or 4-vinylpyridine) are utilized to manganese nanoparticles to make a nano-iniferter, which complex with the template molecules in the conventional was casted onto the surface of a pencil graphite electrode molecular imprinting system which can, however, (PGE) and subsequently used to initiate the surface result in many different structures in the case of protein imprinting of PSA with itaconic acid as the functional templates due to their multifunctional characteristics. To monomer and ethylene glycol dimethacrylate (EGDMA) as overcome this problem, the scaffold imprinting approach the crosslinker in dimethyl sulfoxide at 50°C. The resulting was developed by using a macromolecular chain with PSA-sensor displayed good analytical performance for the some immobilized functional groups (or namely scaffold detection of PSA by square wave and differential pulse polymer) to interact with the large template molecules stripping voltammetric (SWSV and DPSV) techniques, instead of using small functional monomers, which has with a detection limit as low as 0.25 pg L-1 (for SWSV) proven to lead to better imprinted binding sites for such and 3.04 pg L-1 (for DPSV) at a signal to noise ratio of 3, large templates. The protein-MIPs have been readily respectively. In addition, the sensor was also applied for prepared via this strategy through the first synthesis of the easy determination of PSA in human blood serum, some scaffold polymers with required functional groups urine, and forensic samples with high selectivity and via ATRP, the formation of preassembled complexes sensitivity. These appealing characteristics make this between these scaffold polymers and protein templates, MIP-based electrochemical sensor a highly promising and their subsequent crosslinking in the presence of some substitute for the commercially available ELISA kits in appropriate functional monomers and crosslinkers. Mi PSA determination. and coworkers described the first introduction of ATRP 40 H. Zhang into the synthesis of protein-imprinted polymers with in Tris-HCl buffer (pH 7.5). After removing the template, cloned pCyP18 as the template by using this strategy binding sites that were complementary to the target (Fig. 3) [45]. In their work, a scaffold polymer (or “memory protein in size, shape and the position of recognition polymer chain”) with a polymerization degree of 28 and groups were generated. The obtained MIP was used randomly distributed carboxylic acid groups and C=C to adsorb authentic pCyP18 from cell extracts, and its groups was prepared via the combined use of ATRP and proportional content was enriched 300 times. post-modification. The presence of double bond and Recently, Ulbricht and coworkers reported the carboxyl functionalities in the polymer chains facilitated preparation of protein-MIP hydrogel films by combining both cross-linking and hydrogen-bonding abilities with surface imprinting and scaffold imprinting approaches the protein template, respectively. The cloned pCyP18 was in a two-step process [46,47]. The imprinting of then selectively assembled with scaffold polymer chains lysozyme was carried on the track-etched polyethylene and the resulting assemblies (together with a mixture of terephthalate (PET) membrane surface with immobilized AAm and MBA) were adsorbed by the porous polymeric ATRP initiating groups (i.e., aliphatic C-Br groups) (Fig. 4) beads, and immobilized by crosslinking polymerization [46]. Scaffold polymer (i.e., poly(MAA) or PMAA) chains

Figure 3. Strategy for the synthesis of protein-MIP microspheres by ATRP. Reproduced with permission of Elsevier B.V. from [45].

Figure 4. Protein imprinting via two step grafting using surface-initiated-ATRP and UV-initiated grafting/crosslinking copolymerization on the track-etched PET membrane surface. Reproduced by permission of The Royal Society of Chemistry from [46]. Recent Advances in Macromolecularly Imprinted Polymers by Controlled Radical Polymerization Techniques 41 were prepared on the PET surface firstly through the and they proved to exhibit higher rebinding capacity synthesis of poly(tert-butyl methacrylate) brushes via than the non-imprinted polymer films in a wide range of surface-initiated ATRP and their subsequent hydrolysis, Mb concentrations and a selectivity coefficient of 3.15 for which were allowed to assemble with the template protein the template Mb. Takeuchi and co-workers described the lysozyme to form stable complexes. A lysozyme-imprinted synthesis of ribonuclease A (RNase)-imprinted ultrathin polyacrylamide (PAAm) hydrogel layer was then generated polymer films on the gold surfaces of either quartz crystal around the scaffold/protein complexes via UV-initiated microbalance (QCM) or surface plasmon resonance (SPR) surface grafting/crosslinking copolymerization and the sensor chips via the surface-initiated activators generated subsequent removal of lysozyme. This two step approach by electron transfer (AGET) ATRP of acrylic acid, AAm, and allows the independent optimization of the scaffold chain MBA in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid length, UV grafting/crosslinking time, and crosslinking (HEPES)-sodium hydroxide buffer (pH 7.4) in the presence degree of the PAAm-based hydrogel, thus leading to of RNase [51]. An optimum thickness of 15−30 nm was lysozyme-imprinted hydrogel layer with high template obtained for RNase-MIP films to show selective binding selectivity and binding capacity. Following this work, toward the template. In particular, the high effectiveness the same group further extended the above approach of CRP on the protein recognition ability of the resulting to the imprinting of a large biomacromolecule (i.e. the protein-MIPs has been demonstrated in comparison to the antibody immunoglobulin (IgG) [47]. The resulting IgG- traditional free radical polymerization. In the above cases, imprinted hydrogel layer on track-etched PET surface was the potential advantages of these MIP films prepared by demonstrated to be suited for selectively capturing the the surface-initiated ATRP include the possibility of target antibody from a complex mixture. tailoring the resulting materials to have high capacities In the second strategy, ATRP was directly applied to by growing thicker films or high binding efficiencies by prepare protein-MIPs in one step [48-53]. The first such growing thinner films due to the controllable nature of example was reported by Qiu and co-workers [48]. In ATRP. their work, surface-initiated ATRP was used to prepare Recently, some advanced protein-imprinted polymers lysozyme-MIP films on the surfaces of magnetic particles have also been successfully prepared by the combined in aqueous media under mild reaction conditions in use of the surface-initiated ATRP technique and protein the absence of heating and ultraviolet irradiation, surface anchoring strategy (i.e. immobilization of with lysozyme, NIPAAm, AAm, and MBA as the protein templates onto the substrate surfaces prior to the template, functional monomer, assistant monomer, and molecular imprinting polymerization) [52,53]. Our group crosslinker, respectively. The obtained magnetic core- reported a facile, general, and highly efficient approach to shell MIP particles exhibited higher specific recognition preparing uniform core-shell protein-MIP particles with and selectivity toward the template both in a mixture high enzyme inhibition potency using surface-initiated of standard proteins and in real samples and their ATRP with the aid of enzyme surface anchoring strategy superparamagnetic susceptibility allows the separation (Figure 5) [52]. It involved firstly the synthesis of uniform, process to be completed in a short time period by applying highly cross-linked, and “living” polymer microspheres an external magnetic field. In addition, the same group with both ATRP initiating groups and epoxy groups on also prepared another core-shell magnetic protein-MIP their surfaces via our recently developed atom transfer with good protein recognition ability using a similar radical precipitation polymerization (ATRPP) technique approach, but with a larger protein (i.e. bovine serum [54,55], their surface modification to introduce strong albumin (BSA, MW = 66.0 kDa)) as the template (where enzyme-anchoring groups (i.e. benzamidine groups), NIPAAm was used as the functional monomer, and a the immobilization of enzymes (trypsin or kallikrein) basic functional monomer N-[3-(dimethylamino)propyl] onto the polymer microspheres by their interaction with methacrylamide was used as the assistant monomer) the anchoring groups, and the subsequent controlled [49]. Similarly, Caykara and co-workers applied surface- surface molecular imprinting by surface-initiated ATRP initiated ATRP to the construction of myoglobin (Mb)- with AAm and/or 2-hydroxyethyl methacrylate (HEMA) imprinted polymer films onto silica wafer surfaces as the functional monomer and MBA as the crosslinker by using Mb as the template, HEMA as the functional in aqueous buffer solutions at ambient temperature. monomer, EGDMA as the crosslinker, and a mixture of The thickness of the enzyme-imprinted surface layers methanol and water (1:1 v/v) as the solvent [50]. Ultrathin of the core-shell MIP microspheres had a significant MIP films with controlled thickness and smooth surfaces influence on their binding properties, and only those were readily obtained under mild reaction conditions, with their thickness comparable with the diameters of 42 H. Zhang

Figure 5. Schematic protocol for the synthesis of enzyme-imprinted core-shell polymer microspheres with high inhibition potency by the controlled surface molecular imprinting approach: “living” polymer microspheres with surface-bound epoxy groups (a), enzyme-anchoring groups (b), or enzymes (c) and the core-shell polymer microspheres with either immobilized enzymes (d) or enzyme-imprinted pockets (e) on the surface layers. Reproduced with permission from [52]. Copyright 2013 American Chemical Society. the targeted enzymes could afford enzyme-MIPs with first prepared gold (Au)-coated SPR sensor chips with optimal specific bindings. The as-prepared enzyme-MIPs a mixed self-assembled monolayer (SAM) that has both were found to have homogeneous binding sites and high ATRP initiating groups (i.e. aliphatic C-Br groups) and template binding capacities, affinity and selectivity. immobilized GST-π ligand (i.e. L-glutathione (GSH)) via a In addition, they demonstrated much higher enzyme multiple step procedure, which could directly immobilize inhibition potency than the small inhibitor by three GST-π in the PBS buffer (pH 7.4). The SPR chips with orders of magnitude (i.e. the enzyme inhibition constant C-Br groups and immobilized GST-π were then used as of every binding site of the MIP microspheres was about the immobilized ATRP initiator to induce the controlled one-thousandth of that of the small inhibitor), mainly surface-initiated AGET ATRP of AAm, HEMA, and MBAA due to the formation of strong long-range secondary in HEPES buffer at 40°C. The protein-imprinted polymer interactions between enzymes and imprinted pockets. films with high affinity and selectivity toward the target Furthermore, the general applicability of the strategy protein were readily obtained after removal of the was confirmed. Takeuchi and co-workers also performed template protein. The thickness of the MIP films was also precisely controlled molecular imprinting of glutathione- found to have significant influence on their template s-transferase-π (GST-π, a cancer biomarker) by orientated binding selectivity, and a more hydrophilic polymer template immobilization using specific interaction with matrix in the presence of sodium chloride gave more an anchored ligand on a gold substrate (Fig. 6) [53]. They selective binding of GST-π. Recent Advances in Macromolecularly Imprinted Polymers by Controlled Radical Polymerization Techniques 43

Figure 6. Preparation of MIP thin films for glutathione-s-transferase-π (GST-π). Reproduced by permission of The Royal Society of Chemistry from [53].

2.3 Application of RAFT polymerization the binding of control proteins and enhance the specific recognition ability of the MIPs. Zhang and co-workers RAFT polymerization has proven to be one of the most described the facile and efficient synthesis of protein-MIP versatile CRPs because of its applicability to a wide range particles via surface-initiated RAFT polymerization [58]. of monomers (most monomers polymerizable by free It involved firstly the preparation of silica particles with radical methods) and its mild reaction conditions [30]. surface-bound RAFT agent, the adsorption of template The controllability of RAFT polymerization lies in the use protein lysozyme onto the modified silica particles, and of an efficient reversible chain transfer agent (normally the subsequent surface-initiated RAFT copolymerization a dithioester), which results in a fast and dynamic of MAA, HEMA, and MBA. The polymerization was equilibrium between active species (propagating radicals) performed at 40°C in aqueous phase through the and dormant species (thiocarbonylthio-terminated oxidation-reduction initiation. The resulting protein- chains, Scheme 1c) in the polymerization process and imprinted particles showed good recognition selectivity thus controlled polymerization. and rapid mass transfer toward the template protein in Since the first application of RAFT polymerization both protein competitive recognition and real samples, in molecular imprinting [56], many well-defined MIPs but with relatively low binding capacity. In addition, have been prepared via this CRP for a wide range of small their selective capture of the template protein (lysozyme) analytes [22]. In comparison, the imprinting of proteins from a chicken egg white sample was also demonstrated. with RAFT polymerization has been rather limited [57-59]. Furthermore, RAFT polymerization was found to provide Guo and co-workers reported the first preparation of protein-MIP particles with better imprinting effect than protein-MIP microspheres via suspension polymerization those obtained via traditional free radical polymerization. in the presence of a dithioester group-containing Very recently, Zhao and co-workers have demonstrated glycopolymer with bovine serum albumin as the template, a facile approach for the construction of specific and methyl methacrylate (MMA) as the functional monomer, biocompatible fluorescent magnetic antibody-like and EGDMA as the crosslinker at 40°C [57]. The MIP nanoparticles (ANPs) via RAFT polymerization (Fig. 7) microspheres with surface-grafted protected glycopolymer [59]. Inorganic magnetic nanoparticles (Fe3O4@SiO2) with chains and their diameters being around 10−70 μm surface-immobilized azo free radical initiating groups were readily obtained. After alcoholysis of the protected were first prepared, which were then used to initiate glycopolymer, the resulting MIP particles showed the RAFT copolymerization of a mixture of functional improved surface hydrophilicty, which proved to reduce monomers (including N-(3-(dimethylamino) propyl) 44 H. Zhang

Figure 7. (a) Schematic illustration of the one-step synthetic approach for the PEG-modified poly(NIPPAm)-based Fe3O4@SiO2@MIP biocompatible fluorescent antibody-like nanoparticles (ANPs). ACPA is 4,4’-azobis-(4-cyanopentanoic acid). (b) Schematic depiction of in situ sequestration of the target proteins in living cells by using the obtained ANPs. Reproduced by permission of The Royal Society of Chemistry from [59]. methacrylamide, acrylic acid, and NIPPAm), a fluorescent ATRP and RAFT polymerization) has enabled the monomer (fluorescein O-methacrylate), and a crosslinker synthesis of various protein-MIPs with well-defined (MBA) in the presence of the protein template DNase I and structures and improved binding properties toward many a hydrophilic macromolecular RAFT agent (PEG macro- different protein templates. In particular, CRPs have

CTA, average Mn = 10 kDa) under UV light irradiation. The proven to possess highly reproducible polymer surface as-prepared ANPs had a highly compact structure with an chemistry and they are very versatile in the preparation of overall size of 83 ± 5 nm diameter and showed excellent flat surface- or spherical particle-supported uniform and aqueous dispersion stability, negligible undesirable defects-free protein-MIP films with controllable thickness non-specific interactions with other biomolecules, and at the nanoscale (thus leading to easy removal of protein low cytotoxicity. Moreover, they can be readily taken up templates and their rapid binding kinetics as well as good by living cells and selectively sequester target proteins quality protein-MIP films (e.g. with high stability or long within the cells. This approach may be readily extended lifetime) for SPR sensing), which makes them highly to generate ANPs for other proteins of interest and provide useful for the design of sensors using protein-MIP films as useful tools for related biological research and biomedical recognition materials. Moreover, the protein-MIP micro- applications. or nanoparticles prepared via CRPs have also shown great potential in biomedical applications as promising biomedicine [52,59]. Furthermore, since CRPs allow the 3 Conclusions controlled synthesis of MIPs with tailor-made structures, it is now possible for us to study in detail the structure- This mini-review has presented a detailed summary of the property relationship of the protein-MIPs, which will progress in the preparation of protein-MIPs by CRPs, since certainly help us understand more deeply the protein- the first report in 2006 to the middle of 2015. As evidenced MIPs and provide important knowledge for the future by the discussed literature examples, the controllability of protein-MIPs’ development. Although the use of NMP CRPs (including iniferter-induced radical polymerization, in the imprinting of proteins has not been disclosed yet Recent Advances in Macromolecularly Imprinted Polymers by Controlled Radical Polymerization Techniques 45

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