Silica nanoparticle supported molecularly imprinted polymer layers with varied degrees of crosslinking for lysozyme recognition

Surface imprinting over nanosized support materials is particularly suitable for protein templates, considering the problems with mass transfer limitation and low binding capacity. Previously we have demonstrated a strategy for surface protein imprinting over vinyl-modified silica nanopartiles with lysozyme as a model template by polymerization in high-dilution monomer solution to prevent macrogelation. Herein, the synthesis process was further studied toward enhancement of the imprinting performance by examining the effect of several synthesis conditions. Interestingly, the feed crosslinking degree was found to have a great impact on the thickness of the formed imprinting polymer layers and the recognition properties of the resulting imprinted materials. The imprinted particles with a crosslinking degree up to 50% showed the best imprinting effect. The imprinting factor achieved 2.89 and the specific binding reached 23.3 mg g⁻¹, which are greatly increased compared to those of the lowly crosslinked imprinted materials reported previously. Moreover, the relatively high crosslinking degree led to no significant retarding of the binding kinetics to the imprinted particles, and the saturated adsorption was reached within 10 min. Therefore, this may be a promising method for protein imprinting.     

Selective recognition of ovalbumin using a molecularly imprinted polymer

Micro-contact imprinting has been used to form thin-film molecular imprints of ovalbumin (OVA) in polymers supported on glass slides. Thermocalorimetric data was used to optimise the choice of functional monomer and cross-linker to maximise selectivity and minimise non-specific recognition.A polymer comprising polyethyleneglycol 400 dimethacrylate (95 vol.%) and methacrylic acid (5 vol.%) showed both maximum recognition for OVA when made as a molecularly imprinted polymer (MIP), and minimal recognition when made as a non-imprinted, i.e. control polymer. OVA rebinding to the molecularly imprinted polymer, from a buffered 2 µM OVA solution, was 1.55 × 10^{− 11} mol cm^{− 2}, while the control polymer showed 10-fold less re-binding, i.e. 0.154 × 10^{− 11} mol cm^{− 2}.Experiments in which human serum albumin (HSA), conalbumin, ovomucoid or lysozyme, were re-bound to the polymers, either as single proteins or in competition with OVA, showed them to have low affinity for the polymer formulation used. Of the competing proteins examined, in non-competitive binding experiments, HSA showed the greatest affinity 0.45 × 10^{− 11} mol cm^{− 2} for the OVA imprinted polymer. In two protein competition experiments, i.e. with OVA and a competing protein present at equal concentrations (2 µM), OVA binding to the OVA imprinted polymer was in all cases significantly greater than that of the competitor.     

Bio-synthesis of molecularly imprinted membrane with photo-regeneration availability for selective separation applications

Since the in-depth exploration of molecularly imprinted membranes (MIMs), compatibility between high selectivity and superior regeneration has been always the focus. Here we propose, based on biomimetic self-polymerization of dopamine, highly specific, self-cleaning, and interfacial-stable g-C₃N₄/Ag₃PO₄/PDA-composited molecularly imprinted membranes (C/A/D-MIMs) for enhanced treatment of fit-for-purpose water. Ciprofloxacin (CFX) was selected as a representative target because of its potential toxic effect on the microorganism, which will inevitably weaken the traditional biological-based water treatment. Besides superior antifouling performance, remarkable permselectivity (β_NFX/CFX = 1.65, β_EFX/CFX = 1.90, and β_OFX/CFX = 2.08) and flux regeneration rate (84.4% after photo-regeneration) could be ascribed to (i) effective formation of molecularly imprinted sites by self-polymerization of dopamine, (ii) synergism of polydopamine in constructing Z-scheme heterojunction configuration, as well as (iii) bio-adhesion of polydopamine in coupling photocatalyst and MIMs. Achievements in the present work will promote the development of highly specific and self-cleaning MIMs with considerable stability and applications in specific and selective separation.     

Preparation of bovine hemoglobin-imprinted polymer beads via the photografting surface-modified method

Molecularly imprinted polymers (MIPs), based on photografting surface-modified polystyrene beads as matrices, were prepared with acrylamide as the functional monomer, bovine hemoglobin as the template molecule and N, N′-methylene bisacrylamide as the crosslinker in a phosphate buffer. The results of IR, scanning electron microscope (SEM) and elemental analyses demonstrated the formation of a grafting polymer layer on the polystyrene-bead surface. Subsequent removal of the template left behind cavities on the surface of the polymer matrix with a shape and an arrangement of functional groups having complementary binding sites with the original template molecule. The adsorption studies showed that the imprinted polymers have a good adsorption capacity and specific recognition for bovine hemoglobin as the template molecule. Our results demonstrated that the polymer prepared via the photografting surface-modified method exhibited better selectivity for the template. Attempts to employ the new method in molecular imprinting techniques may introduce new applications for MIPs and facilitate probable protein separation and purification. __________ Translated from Chemical Journal of Chinese Universities, 2008, 29(1): 64–70     

Synthesis and evaluation of new propazine-imprinted polymer formats for use as stationary phases in liquid chromatography

Silica particles have been used as supports for the preparation of three different propazine-imprinted polymer formats. First format refers to grafting of thin films of molecularly imprinted polymers (MIPs) using an immobilised iniferter-type initiator (inif-MIP). The other two new formats were obtained by complete filling of the silica pores with the appropriate polymerisation mixture leading to a silica-MIP composite material (c-MIP) followed by the dissolution of the silica matrix resulting in spherical MIP beads (dis-MIP). These techniques offer a mean of fine-tuning the particle morphology of the resulting MIP particles leading to enhanced capacity in chromatographic applications. Porous silica (specific surface area S = 380 m² g⁻¹, particle size p_s = 10 μm, pore volume V_p = 1.083 ml g⁻¹ and pore diameter d_p = 10.5 nm), methacrylic acid and ethylenglycol dimethacrylate were used for the preparation of the materials. All the MIP formats imprinted with propazine have been characterised by elemental analysis, FT-IR spectroscopy, nitrogen adsorption and scanning electron microscopy. Further, the materials were assessed as stationary phases in HPLC. Capacity factors, imprinting factors and theoretical plate numbers were calculated for propazine and other related triazines in order to compare the chromatographic properties of the three different stationary phases. For the inif-MIPs the column efficiency depended strongly on the amount of grafted polymer. Thus, only the polymers grafted as thin films of ca. 1.3 nm average thickness show imprinting effects and the highest column efficiency giving plate numbers (N) of 1600 m⁻¹ for the imprinted propazine. The performance of the c-MIP stationary phase decreases as result of the complete pore filling after polymerisation and increases again after the removal of the silica matrix due to a better mass transfer in the porous mirror-image resulting polymer. From this study can be concluded that the inif-MIP shows the best efficiency for use as stationary phase in HPLC for the separation of triazinic herbicides.     

Surface imprinted thin polymer film systems with selective recognition for bovine serum albumin

Molecularly imprinted polymers are synthetic antibody mimics formed by the crosslinking of organic or inorganic polymers in the presence of an analyte which yields recognitive polymer networks with specific binding pockets for that biomolecule. Surface imprinted polymers were synthesized via a novel technique for the specific recognition of bovine serum albumin (BSA). Thin films of recognitive networks based on 2-(dimethylamino)ethyl methacrylate (DMAEMA) as the functional monomer and varying amounts of either N,N′-methylenebisacrylamide (MBA) or poly(ethylene glycol) (400) dimethacrylate (PEG400DMA) as the crosslinking agent were synthesized via UV free-radical polymerization and characterized. A clear and reproducible increase in recognition of the template BSA was demonstrated for these systems at 1.6-2.5 times more BSA recognized by the MIP sample relative to the control polymers. Additionally, these polymers exhibited selective recognition of the template relative to competing proteins with up to 2.9 times more BSA adsorbed than either glucose oxidase or bovine hemoglobin. These synthetic antibody mimics hold significant promise as the next generation of robust recognition elements in a wide range of bioassay and biosensor applications.     

苯丙氨酸衍生物分子印迹聚合物的制备及手性拆分研究

以分子印迹技术合成分子印迹聚合物，作为手性色谱柱的固定相来拆分苯丙氨酸衍生物的对映异构体。采用了新型的交联剂及光引发剂。模板分子和可聚合的功能单体形成配合物是分子印迹聚合物必不可少的条件。模板分子与功能单体的比例为1:4时，获得了良好的分离效果，分离因子α为1.69。光聚合方式合成的分子印迹聚合物比热聚合方式合成的拆分能力要高。且聚合的温度越低，聚合物分离效果越好。     

Halloysite‐based dopamine‐imprinted polymer for selective protein capture

We describe a facile, general, and highly efficient approach to obtain polydopamine‐coated molecularly imprinted polymer based on halloysite nanotubes for bovine serum albumin. The method combined surface molecular imprinting and one‐step immobilized template technique. Hierarchically structured polymer was prepared in physiological conditions adopting dopamine as functional monomer. A thin layer of polydopamine can be coated on the surface of amino‐modified halloysite nanotubes by self‐polymerization, and the thickness of the imprinted shells can be controlled by the mass ratio of matrix and dopamine. The polymer was characterized by Fourier transform infrared spectrometry, transmission electron microscopy, and thermogravimetric analysis. The prepared material showed high binding capacity (45.4 mg/g) and specific recognition behavior toward the template protein. In addition, stability and regeneration analyses indicated that the imprinted polymer exhibited excellent reusability (relative standard deviation < 9% for batch‐to‐batch evaluation). Therefore, the developed polymer is effective for protein recognition and separation.     

Noncovalently galactose imprinted polymer for the recognition of different saccharides

Molecularly imprinted polymers (MIPs) represent a new class of materials possessing high selectivity and affinity for the target molecule. The main goal of this study was to prepare a galactose imprinted polymer and its potential application for the recognition of different saccharides. The selectivity of galactose imprinted polymer for several saccharides; glucose, mannose, fructose, maltose, lactose, sucrose and raffinose was investigated. Macroporous polymer was prepared utilizing ethyleneglycoldimethacrylate as a crosslinking agent, in the presence of galactose as a template molecule with acrylamide as a functional monomer. After the synthesis of polymer, galactose was removed by methanol:acetic acid washing. The selectivity of galactose imprinted polymer for other saccharides was utilized by batch rebinding assay. The arrangement of functional groups within cavities versus shape selectivity is discussed. The results showed that, the orientation of the functional groups was the dominating factor for the selectivity of galactose imprinted polymer. The dissociation constants of polymer were determined by Scatchard analysis.     

Separation of proteins in a multicompartment electrolyzer with chambers defined by a bed of gel beads

Multicompartment electrolyzers (MEs) with isoelectric membranes were introduced in 1989 for purifying proteins in an electric field. At the basis of ME technology there are membranes consisting of cross-linked copolymers of acrylamide and acrylamido monomers bearing protolytic groups. The technology employed for casting the membranes is an extension of the isoelectric focusing in immobilized pH gradient technique for which specific acrylamido monomers, known with the trade name of Immobiline, have been developed. However, the use of continuous membranes presents several disadvantages. Due to the mechanical characteristics of polyacrylamide, the gel must physically adhere onto a rigid support, which prevents it from collapsing. The support must have a highly porous structure in order to be permeable to proteins. The mechanical fragility of the membranes is one of the main problems that hinders the industrial scale application of ME separators. In order to overcome this problem, we propose to substitute the continuous membranes with a bed of gel beads of identical comonomer composition, obtained by an inverse emulsion polymerization process.     

Adaptation of the molecular imprinted polymers towards polar environment

A new simple method for the post-polymerisation treatment of molecularly imprinted polymers was proposed. A layer of mineral oil was deposited onto the surface of the polymer in order to create a hydrophobic environment in the binding sites and to improve the recognition properties of the polymer in polar solvents. The testing of polymers performed in acetonitrile showed that the modified polymers possessed significantly increased selectivity as compared with non-treated ones. The three-fold improvement of recognition of the template (cocaine) was achieved; the same time, for non-specific molecule (morphine) the improvement was only 1.3 times. The investigation of the stability of mineral oil coating on the polymer surface suggested that the effect produced is stable over a long period of time. This approach could be used to broaden the range of experimental conditions where molecularly imprinted polymers can perform successfully.     

Synthesis of polystyrene beads loaded with dual luminophors for self-referenced oxygen sensing

Dispersion polymerization has been successfully applied to synthesize monodisperse polystyrene beads loaded with SiOEP and PtOEP for self-referenced oxygen sensing. The polystyrene beads became larger in size as the concentration of initiator was increased due to the reduction of primary particles precipitated from the polymerization medium. The dual luminophors showed similar absorption spectra but two distinctive emission spectra with peaks at 580 and 650 nm for SiOEP and PtOEP, respectively. While the emission of SiOEP exhibited no response to oxygen, the luminescence intensity of PtOEP was monotonically dependent on the concentration of oxygen. From the Stern-Volmer plot, we observed a linear correlation between the intensity ratio of SiOEP at 580 nm to PtOEP at 650 nm and the concentration of oxygen, which could be used to reliably monitor the partial pressure of oxygen in a system.     

Molecularly imprinted polymer for metsulfuron-methyl and its binding characteristics for sulfonylurea herbicides

A molecularly imprinted polymer (MIP) was prepared using metsulfuron-methyl (MSM) as the template molecule. A combinatorial protocol has been employed to optimize the polymer in terms of the kind and relative amounts of functional and cross-linking monomers. A copolymer of 2-(trifluoromethyl)acrylic acid (TFMAA) and divinylbenzene (DVB) showed the highest binding capacity for MSM. The binding characteristics of the imprinted polymers and MSM were evaluated in various solvents using equilibrium binding experiments. The results showed that the MIP binds MSM only in dichloromethane, which was used as the porogen during polymerization. Scatchard plot analysis revealed that two classes of binding sites were formed in the imprinted polymer with dissociation constants of 32.3 μmol l⁻¹ and 1.7 mmol l⁻¹, respectively. The specificity of the imprinted polymer was investigated by binding assays using MSM and other structurally related sulfonylurea herbicides. The results indicated that the imprinted polymer showed a marked selectivity for MSM.     

Molecularly imprinted supermacroporous cryogels for cytochrome c recognition

Molecular imprinting is an attractive biomimetic approach that creates specific recognition sites for the shape and functional group arrangement to template molecules. The purpose of this study is to prepare cytochrome c-imprinted poly(hydroxyethyl methacrylate) (PHEMA)-based supermacroporous cryogel which can be used for the separation of cytochrome c from protein mixtures. N-Methacryloyl-(L)-histidinemethylester (MAH) was used as the metal-coordinating monomer. In the first step, Cu(2+) was complexed with MAH, and the cytochrome c imprinted PHEMA (MIP) cryogel was prepared by free radical cryopolymerization initiated by N,N,N',N'-tetramethylene diamine at -12°C. After polymerization is completed, the template cytochrome c molecules were removed from the MIP cryogel using 0.5 M NaCl solution. The maximum cytochrome c binding amount was 126 mg/g polymer. Selective binding studies were performed in the presence of lysozyme and bovine serum albumin. The relative selectivity coefficients of MIP cryogel for cytochrome c/lysozyme and cytochrome c/bovine serum albumin were 1.7 and 5.2 times greater than those of the non-imprinted PHEMA cryogel, respectively. The selectivity of MIP cryogel for cytochrome c was also confirmed with fast protein liquid chromatography. The MIP cryogel could be used many times with no remarkable decrease in cytochrome c binding capacity.     

Monodisperse polymer beads as packing material for high-performance liquid chromotography: Effect of divinylbenzene content on the porous and chromatographic properties of poly(styrene-co-divinylbenzene) beads prepared in presence of linear polystyrene as a porogen

The effect of concentration of divinylbenzene on pore size distribution and surface areas of micropores, mesopores, and macropores in uniformly sized porous poly(styrene-co-divinylbenzene) beads prepared in the presence of linear polystyrene as a component of the porogenic mixture has been studied. While the total specific surface area was clearly determined by the content of divinylbenzene, the sum of pore volumes for mesopores and macropores as well as their size distribution does not change within a broad range of DVB concentrations. Consequently, the size exclusion chromatography calibration curves are almost identical for all the beads prepared with different percentages of crosslinking monomer. However, the more crosslinked beads have better mechanical and hydrodynamic properties. © 1994 John Wiley & Sons, Inc.     

Selective extraction of antioxidants with molecularly imprinted polymers

Molecular imprinting technology can be used to generate specific artificial polymeric receptors, i.e., high affinity stationary phases, as already shown for peptides and many other food ingredients. In this work, polymers have been molecularly imprinted with three exemplary antioxidants, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG). Results of chromatographic evaluations are presented, demonstrating the specificity of the imprinted polymers (MIP) compared to non-specific control polymers (CP). For a BHA imprinted polymer and the respective analyte BHA a separation factor of α=k_BHA-MIP′/k_CP′=1.12 was found, whereas for the BHT-MIP a higher selectivity was determined with α=k_BHT-MIP′/k_CP′=1.47. Using the PG selective MIP for PG as analyte a value of α=k_PG-MIP′/k_CP′=1.24 could be achieved. Furthermore, results of extraction procedures based on MIP and CP phases are presented, demonstrating for instance a higher selectivity of the BHA-MIP in comparison with its CP when using methanol or acetonitrile as analyte solvent, and of the PG-MIP when performing the extraction in acetonitrile.     

Selectivity and recovery performance of phosphate-selective molecularly imprinted polymer

A phosphate-selective molecularly imprinted polymer was prepared using 1-allyl-2-thiourea as a functional monomer, and the binding ability and selectivity of the polymer were evaluated. The imprinted polymer showed high binding ability to and selectivity for phosphate in aqueous media. The recoverability of phosphate from the imprinted polymer was also examined, and nearly 70% of highly concentrated phosphate could be recovered.     

Synthesis and chromatographic evaluation of molecularly imprinted polymers prepared by the substructure approach for the class-selective recognition of glucuronides

Two series of molecularly imprinted polymers (MIPs) for the class-selective recognition of glucuronides have been prepared by using lipophilic substructures of the target analyte as template molecule and potent host monomers against oxyanions, that are expected to establish a strong stoichiometric interaction with the single carboxylic group of the template. The polymers were tested as stationary phases in liquid chromatography for specific recognition. A preliminary investigation of the imprinting properties of eleven MIPs was carried out, by comparing the retention time of the template and of structurally related compounds on the MIP column with that on the corresponding non-imprinted polymer (NIP). The two polymers showing the best performance were selected to further test cotinine, mycophenolic acid, testosterone and their respective glucuronides as model compounds. The high specificity obtained against glucuronides and the different chemical structure of the parent drug make the two MIPs class-selective imprinted receptors, also suitable for SPE application.     

Preparation and evaluation of a macroporous molecularly imprinted hybrid silica monolithic column for recognition of proteins by high performance liquid chromatography

A novel type of macroporous molecularly imprinted hybrid silica monolithic column was first developed for recognition of proteins. The macroporous silica-based monolithic skeleton was synthesized in a 4.6 mm i.d. stainless steel column by a mild sol–gel process with methyltrimethoxysilane (MTMS) as a sole precursor, and then vinyl groups were introduced onto the surface of the silica skeleton by chemical modification of γ-methacryloxypropyltrimethoxysilane (γ-MAPS). Subsequently, the molecularly imprinted polymer (MIP) coating was copolymerized and anchored onto the surface of the silica monolith. Bovine serum albumin (BSA) and lysozyme (Lyz), which differ greatly in molecular size, isoelectric point, and charge, were representatively selected for imprinted templates to evaluate recognition property of the hybrid silica-based MIP monolith. Some important factors, such as template–monomer molar ratio, total monomer concentration and crosslinking density, were systematically investigated. Under the optimum conditions, the obtained hybrid silica-based MIP monolith showed higher binding affinity for template than its corresponding non-imprinted (NIP) monolith. The imprinted factor (IF) for BSA and Lyz reached 9.07 and 6.52, respectively. Moreover, the hybrid silica-based MIP monolith displayed favorable binding characteristics for template over competitive protein. Compared with the imprinted silica beads for stationary phase and in situ organic polymer-based hydrogel MIP monolith, the hybrid silica MIP monolith exhibited higher recognition, stability and lifetime.