Huiqi Zhang

Narrowly Dispersed Hydrophilic Molecularly Imprinted Polymer Nanoparticles for Efficient Molecular Recognition in Real Aqueous Samples Including River Water, Milk, and Bovine Serum

Molecular imprinting has proven to be a versatile approach to the preparation of synthetic receptors with tailor-made recognition sites. The ultimate goal of molecular imprinting is to generate molecularly imprinted polymers (MIPs) with affinity and specificity approaching those of the biological receptors so that they can eventually replace such biological entities in real applications. However, previously developed MIPs that target small organic molecules are normally only compatible with organic solvents, and they mostly fail to show specific template binding in aqueous solutions (whereas peptideor protein-imprinted polymers are intrinsically water-compatible), which significantly limits their practical application in such areas as molecularly imprinted sorbent assays and biomimetic sensors. Despite some progress made in the development of MIPs applicable for analyte detection in relatively simple aqueous media, such as pure water, surfactant-containing water, aqueous buffer solutions (mostly containing an organic solvent), and beer or solutions that mimic alcoholic beverages, the design of MIPs directly capable of specifically recognizing the targeted small organic molecules in real biological samples remains a formidable challenge owing to the complex nature of the sample matrices. Herein, we report the efficient synthesis of narrowly dispersed hydrophilic MIP nanoparticles with excellent specific molecular-recognition ability in real aqueous solutions, including river water and biological samples (both diluted and undiluted milk and bovine serum). Reversible addition–fragmentation chain-transfer (RAFT) precipitation polymerization (RAFTPP) mediated by hydrophilic macromolecular chain-transfer agents (macro-CTAs) provided for the first time narrowly dispersed highly cross-linked MIP (or polymer) nanoparticles with surface-grafted hydrophilic polymer brushes in a facile one-pot approach (Figure 1). The hydrophilic polymer brushes on the MIP nanoparticles not only significantly improved their surface hydrophilicity and led to their water compatibility, but they also acted as a protective layer to prevent proteins in the biological samples from accumulating on the nanoparticle surface and blocking the imprinting sites and thus enabled the MIP nanoparticles to function properly in such complex matrices. Although studies on the use of MIPs in biological media for specific molecular recognition have been disclosed, either diluted biological solutions (containing 40 vol% of a mixture of ethanol/water (1:1 v/v) and a phosphate buffer) were used or the targeted molecule was a highly water soluble 26 amino acid peptide (i.e., the bee toxin melittin). In this context, there have been many reports on the MIP-based solid-phase extraction of target analytes in real matrices; however, the selectivity of the MIPs in this case is controlled by the choice of solvents in the extraction procedure. The direct use of MIPs in real matrices is more difficult, as the conditions used are mainly fixed by the nature of the sample. To our knowledge, we report herein the first successful example of the synthesis of MIP nanoparticles that can be used directly in undiluted biological samples for the efficient specific recognition of small organic molecules. This finding is a major breakthrough for molecular-imprinting technology, since it opens the door to the facile preparation of nanoscale MIPs (with good dispersion and outstanding performance in real aqueous samples) that are very attractive synthetic substitutes for biological receptors in bioanalytical applications and many other fields. Figure 1. Preparation and characterization of MIP nanoparticles. a) Chemical structures of the RAFT agents used: hydrophilic macroCTAs and CDB. b) Schematic protocol for the one-pot preparation by RAFTPP of MIP nanoparticles compatible with real aqueous solutions. c) Characterization of MIP nanoparticles (Mn,NMR of PHEMA brushes: 4800) by SEM and DLS.

Efficient One-Pot Synthesis of Water-Compatible Molecularly Imprinted Polymer Microspheres by Facile RAFT Precipitation Polymerization**

Molecular imprinting is a versatile and straightforward method for the preparation of polymer receptors with tailor-made recognition sites. Despite the tremendous progress made in this field, many challenges still remain to be addressed. In particular, it has been shown that the presently developed molecularly imprinted polymers (MIPs) are normally only organic solvent compatible and they mostly fail to show specific template bindings in pure aqueous solutions, thus significantly limiting their practical applications in the field of biotechnology. Although some approaches, which either use specifically designed functional monomers or apply the conventional imprinting protocol, have been developed for the preparation of MIPs with molecular recognition ability under aqueous conditions, versatile approaches for the preparation of MIPs that are applicable in pure aqueous environments are still rare. Herein, we report a new and efficient one-pot approach to obtain pure-water-compatible and narrowly dispersed MIP microspheres with surface-grafted hydrophilic polymer brushes by facile reversible addition/fragmentation chaintransfer (RAFT) precipitation polymerization (RAFTPP), mediated by hydrophilic macromolecular chain-transfer agents (Macro-CTAs; Scheme 1). The presence of hydrophilic polymer brushes on MIP microspheres significantly improved their surface hydrophilicity and dramatically reduced their hydrophobic interactions with template molecules in pure aqueous media, thus leading to their water compatibility. The easy availability of many different hydrophilic Macro-CTAs (by either RAFT polymerization of hydrophilic monomers or hydrophilic polymer end group modification), together with the versatility of RAFTPP for the controlled preparation of MIP microspheres, makes this strategy highly applicable for the design of hydrophilic and water-compatible MIPs. Two strategies have been developed for the synthesis of water-compatibleMIPs by improving their surface hydrophilicity; these strategies involve the use of a hydrophilic comonomer, functional monomer, or crosslinker in the molecular imprinting process, and the postmodification of the preformed MIPs. Although simple in principle, the former strategy either requires time-consuming optimization of MIP formulation components or can only be applied in some special systems. In comparison, the latter strategy, which involves the surface grafting of hydrophilic polymer layers, has proven highly attractive because it not only significantly improves the MIPs surface hydrophilicity, but also provides a protective layer to prevent protein molecules from blocking their imprinting sites in biological solutions. Very recently, we have successfully prepared pure-water-compatible MIP microspheres by the controlled grafting of hydrophilic polymer layers onto the preformed MIP particles. Compared with this two-step approach, the new strategy presented herein allows the more efficient controlled synthesis of pure-water-compatible MIP microspheres with surface-grafted hydrophilic polymer brushes by a one-pot RAFTPP method. To show proof-of-principle for our strategy, a model noncovalent molecular imprinting system was chosen because Scheme 1. Chemical structures of the utilized RAFT agents (including hydrophilic Macro-CTAs and CDB) and the schematic protocol for the one-pot preparation of water-compatible MIP microspheres by RAFT precipitation polymerization.

An efficient approach to obtaining water-compatible and stimuli-responsive molecularly imprinted polymers by the facile surface-grafting of functional polymer brushes via RAFT polymerization

A new and efficient approach to obtaining molecularly imprinted polymers (MIPs) with both pure water-compatible (i.e., applicable in the pure aqueous environments) and stimuli-responsive binding properties is described, whose proof-of-principle is demonstrated by the facile modification of the preformed MIP microspheres via surface-initiated reversible addition-fragmentation chain transfer (RAFT) polymerization of N-isopropylacrylamide (NIPAAm). The presence of poly(NIPAAm) (PNIPAAm) brushes on the obtained MIP microspheres was confirmed by FT-IR as well as the water dispersion and static contact angle experiments, and some quantitative information including the molecular weights and polydispersities of the grafted polymer brushes, the thickness of the polymer brush layers, and their grafting densities was provided. In addition, the binding properties of the ungrafted and grafted MIPs/NIPs in both methanol/water (4/1, v/v) and pure water solutions were also investigated. The introduction of PNIPAAm brushes onto the MIP microspheres has proven to significantly improve their surface hydrophilicity and impart stimuli-responsive properties to them, leading to their pure water-compatible and thermo-responsive binding properties. The application of the facile surface-grafting approach, together with the versatility of RAFT polymerization and the availability of many different functional monomers, makes the present methodology a general and promising way to prepare water-compatible and stimuli-responsive MIPs for a wide range of templates.

Controlled synthesis of water-compatible molecularly imprinted polymer microspheres with ultrathin hydrophilic polymer shells via surface-initiated reversible addition-fragmentation chain transfer polymerization

The first controlled synthesis of pure water-compatible molecularly imprinted polymer (MIP) microspheres with ultrathin hydrophilic poly(2-hydroxyethyl methacrylate) (PHEMA) shells (including both PHEMA brushes and lightly crosslinked PHEMA hydrogel layer) via surface-initiated reversible addition-fragmentation chain transfer (RAFT) polymerization is described. The presence of ultrathin hydrophilic polymer shells on the MIP microspheres was confirmed by SEM, FT-IR, fluorescent labeling treatment, contact angle studies, and water dispersion stability test, and some quantitative information including the thickness of the grafted hydrophilic polymer layers as well as the molecular weights and polydispersities of the grafted polymer brushes and their grafting densities was provided. The facile surface-grafting of both PHEMA brushes and PHEMA hydrogel layer on the MIP microspheres proved to be highly efficient for improving their surface hydrophilicity and suppressing the hydrophobically driven nonspecific interactions between the MIPs and template molecules, leading to MIPs with pure water-compatible binding properties. The findings presented here not only prove the general applicability of the controlled hydrophilic polymer brushes-grafting approach in obtaining pure water-compatible MIPs, but also largely extend the scope of this versatile surface-grafting approach through the controlled surface-grafting of hydrophilic polymer hydrogel layer onto the MIPs. Moreover, the significant effect of the chain length of the grafted polymer brushes and the presence of crosslinking in the grafted polymer shells on the surface hydrophilicity and water-compatibility of the MIP microspheres was also demonstrated for the first time, which is of great importance for the rational design of water-compatible MIPs by using this controlled surface-grafting approach.

Azobenzene-containing molecularly imprinted polymer microspheres with photoresponsive template binding properties

The first successful preparation of azobenzene (azo)-containing molecularly imprinted polymer (MIP) microspheres with photoresponsive template binding properties is described. A methacrylate azo functional monomer with a pyridine group was used for this purpose, and its good solubility in acetonitrile allowed the implementation of molecular imprinting viaprecipitation polymerization, leading to azo-containing MIP microspheres (number-average diameter = 1.33 μm, polydispersity index = 1.15) with obvious molecular imprinting effects towards the template 2,4-dichlorophenoxyacetic acid (2,4-D), rather fast template rebinding kinetics, and appreciable selectivity over structurally related compounds. The binding association constant Ka and apparent maximum number Nmax for high-affinity sites of the imprinted polymer in the dark environment were determined by Scatchard analysis to be 2.3 × 104 M−1 and 10.0 μmol g−1, respectively. Most importantly, the binding affinity of the imprinted sites in azo-containing MIP microspheres was found to be photoresponsive towards the template, which decreased upon UV light irradiation (as revealed by the resulting lower Ka value for high-affinity sites and reduced specific bindings), whereas it could be recovered during the subsequent thermal (or visible light-induced) back-isomerization. Furthermore, this photoregulation process proved to be highly repeatable under photoswitching conditions.

Efficient one-pot synthesis of hydrophilic and fluorescent molecularly imprinted polymer nanoparticles for direct drug quantification in real biological samples

Efficient one-pot synthesis of hydrophilic and fluorescent molecularly imprinted polymer (MIP) nanoparticles and their application as optical chemosensor for direct drug quantification in real, undiluted biological samples are described. The general principle was demonstrated by preparing tetracycline (Tc, a broad-spectrum antibiotic)-imprinted fluorescent polymer nanoparticles bearing hydrophilic polymer brushes via poly(2-hydroxyethyl methacrylate) (PHEMA) macromolecular chain transfer agent-mediated reversible addition-fragmentation chain transfer (RAFT) precipitation polymerization in the presence of a fluorescent monomer. The introduction of hydrophilic PHEMA brushes and fluorescence labeling onto/into the MIP nanoparticles proved to not only significantly improve their surface hydrophilicity and lead to their obvious specific binding and high selectivity toward Tc in the undiluted bovine serum, but also impart them with strong fluorescent properties. In particular, significant fluorescence quenching was observed upon their binding with Tc in such complex biological milieu, which makes these Tc-MIP nanoparticles useful optical chemosensor with a detection limit of 0.26 μM. Furthermore, such advanced functional MIP nanoparticles-based chemosensor was also successfully utilized for the direct, sensitive, and accurate determination of Tc in another biological medium (i.e., the undiluted pig serum) with average recoveries ranging from 98% to 102%, even in the presence of several interfering drugs.

Efficient synthesis of photoresponsive azobenzene-containing side-chain liquid crystalline polymers with high molecular weights by click chemistry

A new and highly efficient approach to obtaining high molecular weight (up to 303000) azobenzene (azo)-containing side-chain liquid crystalline polymers by copper(I)-catalyzed polymer analogous click chemistry is described. A series of azo compounds with an azido end group and different flexible spacers were successfully attached onto a polymer bearing pendant acetylene groups (i.e., poly(propargyl methacrylate)) in rather high functionalization efficiency (≥97%). The chemical structures, phase transition behaviors, and photoresponsivity of the obtained polymers were characterized, and they were also compared with those of the corresponding low molecular weight azo polymers (prepared via conventional free radical polymerization) to study the effects of the largely different molecular weights on the properties of the polymers. Both the high and low molecular weight azo polymers with a flexible spacer = (CH2)10 showed smectic C liquid crystallinity and an increase in the molecular weight led to a broader range of the liquid crystalline phase, which is positive for potential applications. Furthermore, the highly reversible photoisomerization of the polymer solutions was proven to be hardly affected by the increase of the molecular weights.

Comparative study of the molecularly imprinted polymers prepared by reversible addition–fragmentation chain transfer “bulk” polymerization and traditional radical “bulk” polymerization

Bisphenol A (BPA) and propranolol‐imprinted polymers have been prepared via both reversible addition–fragmentation chain transfer “bulk” polymerization (RAFTBP) and traditional radical “bulk” polymerization (TRBP) under similar reaction conditions, and their equilibrium binding properties were compared in detail for the first time. The chemical compositions, specific surface areas, equilibrium bindings, and selectivity of the obtained molecularly imprinted polymers (MIPs) were systematically characterized. The experimental results showed that the MIPs with molecular imprinting effects and quite fast binding kinetics could be readily prepared via RAFTBP, but they did not show improved template binding properties in comparison with those prepared via TRBP, which is in sharp contrast to many previous reports. This could be attributed to the heavily interrupted equilibrium between the dormant species and active radicals in the RAFT mechanism because of the occurrence of fast gelation during RAFTBP. The findings presented here strongly demonstrates that the application of controlled radical polymerizations (CRPs) in molecular imprinting does not always benefit the binding properties of the resultant MIPs, which is of significant importance for the rational use of CRPs in generating MIPs with improved properties. Copyright

Photoresponsive side-chain liquid crystalline polymers with amide group-substituted azobenzene mesogens: effects of hydrogen bonding, flexible spacers, and terminal tails

The synthesis of a series of new photoresponsive side-chain liquid crystalline polymethacrylates with amide group-substituted azobenzene (azo) mesogens and different length of flexible spacers and terminal tails via conventional free radical polymerization is described. The resulting azo polymers proved to have high thermal stability and good solubility in common organic solvents (e.g., tetrahydrofuran and chloroform). Differential scanning calorimetry, polarizing optical microscopy, and small angle X-ray scattering studies confirmed the presence of obvious enantiotropic smectic C liquid crystalline phases (with a bilayer lamellar structure) for all these polymers. The introduction of an amide group into the azo mesogen led to the formation of strong hydrogen bonding among the side chains of the polymers (as revealed by variable temperature FT-IR), which played a decisive role in forming and stabilizing the liquid crystalline mesophases of the polymers. In addition, the length of the flexible spacers and terminal tails also significantly influenced their phase transition behaviors. Furthermore, the photoresponsivity of the polymer solutions was verified and the effects of the molecular structures of the polymers on their photoresponsive properties were also studied.

Ambient temperature synthesis of narrow or monodisperse, highly cross-linked, and “living” polymer microspheres by atom transfer radical precipitation polymerization

A facile ambient temperature atom transfer radical precipitation polymerization (ATRPP) approach is developed for the efficient one-pot synthesis of narrow or monodisperse, highly cross-linked, and “living” polymer microspheres under mild reaction conditions. The simple introduction of an atom transfer radical polymerization (ATRP) mechanism into a precipitation polymerization system, together with the rational use of polar alcoholic solvents, allows the direct ambient temperature preparation of uniform “living” polymer microspheres with their number-average diameters ranging from 0.36–1.95 μm and their particle size distributions being typically less than 1.01. The polymerization parameters (including the monomer loading, polymerization time, and kind of alcoholic solvent) proved to have a pronounced influence on the yields and morphologies of the polymer microspheres, which makes it very convenient to tailor the particle sizes by tuning the polymerization conditions. The general applicability of ambient temperature ATRPP was demonstrated by its successful application in a range of alcoholic solvents as well as its versatility in the synthesis of a series of uniform copolymer microspheres of different monovinyl functional monomers (4-vinylpyridine, glycidyl methacrylate, methyl methacrylate, and 2-hydroxyethyl methacrylate) with ethylene glycol dimethacrylate. In addition, the “livingness” of the resulting polymer microspheres was confirmed by their direct grafting of hydrophilic polymer brushes via surface-initiated ambient temperature ATRP, leading to advanced functional polymer microspheres with significantly improved surface hydrophilicity.