Surface-modified polystyrene beads as photografting imprinted polymer matrix for chromatographic separation of proteins

A new and facile fabricating method for lysozyme molecularly imprinted polymer beads (lysozyme-MIP beads) in aqueous media was presented. Mesoporous chloromethylated polystyrene beads (MCP beads) containing dithiocarbamate iniferter (initiator transfer agent terminator) were used as supports for the grafting of lysozyme imprinted copolymers with acrylamide and N,N′-methylenebisacrylamide through surface initiated living-radical polymerization (SIP). After the polymerization, a layer of lysozyme-MIP was formed on the MCP beads. The SIP allowed an efficient control of the grafting process and suppressed solution propagation. Therefore, the obtained lysozyme-MIP beads had a large quantity of well-distributed pores on the surface without any visible gel formation in solution and were more advantageous comparing with traditional MIPs which were prepared by traditionally initiated radical polymerization. The obtained composites were characterized by Fourier transform infrared spectroscopy, elemental analysis, nitrogen sorption analysis and scanning electron microscopy. Chromatographic behaviors of the column packed with lysozyme-MIP beads exhibited ability in separating lysozyme from competitive protein (bovine hemoglobin, bovine serum albumin, ovalbumin or cytochrome c) in aqueous mobile phase.     

Preparation of a bipolar membrane by photografting polymerization

A single-sheet bipolar membrane was synthesized via photografting polymerization of an acrylic acid cation exchange layer onto the surface of a commercial homogeneous anion exchange membrane with benzophenone (BP) as the main initiator, diphenyl (2,4,6-trimethylbenzoyl)-phosphine oxide (TPO) and 2-hydroxy-2-methyl-1-phenyl-1-propanone (1173) as coinitiators and divinylbenzene (DVB) or neopentylene glycol diacrylate (NPGDA) as crosslinking agent. It was found that when benzophenone was used as single initiator and the crosslink agent is absent, the grafting degree (D _g) increases with the prolongation of irradiation time. The grafting degree reaches a maximum at 60 seconds reaction time when the crosslinking agent is added, and the grafting degree is higher when using NPGDA instead of DVB as crosslinking agent. The grafting degree increases as the composite initiator system is used. When polymerization was initiated by BP and TPO, and the dosage of NPGDA was 2.5% mol concentration of monomer, the grafting degree reaches 30.1%. __________ Translated from Journal of Beijing Technology and Business University, 2007, 25(1): 15–18 [译自: 北京工商大学学报]     

环丙沙星分子印迹聚合物的合成及识别性能研究

采用分子印迹技术合成了以环丙沙星为印迹分子,以甲基丙烯酸和4-乙烯基吡啶同时为功能单体的分子印迹聚合物。运用平衡结合实验研究了印迹聚合物的吸附特性和选择识别能力。Scatchard分析表明,在所研究的浓度范围内,分子印迹聚合物中形成了两类不同的结合位点。底物选择实验表明,这种聚合物对环丙沙星呈现高的选择结合能力。     

Molecularly imprinted polymers with assistant recognition polymer chains for bovine serum albumin

A new protein molecularly imprinted polymer (MIP) was prepared with grafting polyvinyl alcohol as assistant recognition polymer chains (ARPCs). The ARPCs and acrylamide monomers were interpenetrated and then polymerized on the surface of macroporous acrylate adsorbent spheres. The template BSA was removed by treatment with 2.00 mol L-1 potassium chloride (KCl) solution and the adsorbed proteins were detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 0.150, 0.500, and 2.00 mo...     

Polyacrylamide gel beads for the recognition of staphylococcal enterotoxin B

Protein‐imprinted polyacrylamide gel beads (IPGB) were synthesized via inverse suspension polymerization, using staphylococcal enterotoxin B (SEB) as template. The adsorption capacity of SEB‐IPGB was almost three times as much as that of non‐imprinted gel beads. The Langmuir adsorption models were applied to describe the equilibrium isotherms. The results showed that an equal class of adsorption was formed in the SEB‐IPGB with the maximum adsorption capacity of 8.40 mg SEB/g imprinted beads. The selectivity test of imprinted beads shows that they exhibited good recognition for SEB as compared with the other proteins. The formation of multiple hydrogen bonds and complementary shape between the imprinting cavities and the template proteins would be the two factors that led to the imprinting effect. The obtained SEB‐IPGB would be used as a potential material for protein toxin separation, extraction, and purification. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis of a novel methacrylic monomer iniferter and its application in surface photografting on crosslinked polymer substrates

(Methacryloyl ethylenedioxycarbonyl) benzyl N,N‐diethyldithiocarbamate (HEMA‐E‐In) was synthesized and used as a monomer iniferter to develop a novel, photopatternable grafting technology. This molecule functions as both a methacrylic monomer and a photoiniferter (photoinitiator–transfer agent–terminator). The structure of HEMA‐E‐In was characterized by ¹H NMR, Fourier transform infrared, and ultraviolet–visible spectroscopies. In the presence of the monomer iniferter, methyl methacrylate was polymerized by exposure to 365‐nm ultraviolet radiation, confirming the initiation capability of HEMA‐E‐In. After the copolymerization of HEMA‐E‐In into a methacrylate‐based polymer, attenuated total reflectance Fourier transform infrared spectra revealed that the photoiniferter functionality was present at the surface of this polymeric substrate. Photografting of poly(ethylene glycol) monomethacrylate monomer from the surface caused a significant change in the hydrophobicity of the surface as demonstrated by contact angle measurements. The novel monomer photoiniferter HEMA‐E‐In initiates the polymerization of bulk monomer and provides a reactive functionality that facilitates further initiation and polymer modification by the polymerization of different monomers. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 1885–1891, 2002     

Fabrication of temperature-sensitive imprinted polymer hydrogel

Weakly cross-linked temperature sensitive gels that memorize 4-aminopyridine molecules were designed and synthesized. The polymer gels show special selectivity, good thermo-sensitivity and reusability, which would have an enormous potential of application in drug controlled release systems.     

Preparation of molecularly imprinted polymer microspheres via atom transfer radical precipitation polymerization

The first combined use of atom transfer radical polymerization (ATRP) and precipitation polymerization in the molecular imprinting field is described. The utilized polymerization technique, namely atom transfer radical precipitation polymerization (ATRPP), provides MIP microspheres with obvious molecular imprinting effects towards the template, fast template binding kinetics and an appreciable selectivity over structurally related compounds. The living chain propagation mechanism in ATRPP results in MIP spherical particles with diameters (number‐average diameter D_n ≈ 3 μm) much larger than those prepared via traditional radical precipitation polymerization (TRPP). In addition, the MIP microspheres prepared via ATRPP have also proven to show significantly higher high‐affinity binding site densities on their surfaces than the MIP generated via TRPP, while the binding association constants K_a and apparent maximum numbers N_max of the high‐affinity sites as well as the specific template bindings are almost the same in the two cases. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 3257–3270, 2009     

Chromatographic performance of molecularly imprinted polymers: Core‐shell microspheres by precipitation polymerization and grafted MIP films via iniferter‐modified silica beads

In this work, two different surface imprinting formats have been evaluated using thiabendazole (TBZ) as model template. The first format is a thin film of molecularly imprinted polymer (MIP) grafted from preformed silica particles using an immobilized iniferter‐type initiator (inif‐MIP). The second format is molecularly imprinted polymer microspheres with narrow particle size distribution and core‐shell morphology prepared by precipitation polymerization in a two‐step procedure. For the latter format, polymer microspheres (the core particles) were obtained by precipitation polymerization of divinylbenzene‐80 (DVB‐80) in acetonitrile. Thereafter, the core particles were used as seed particles in the synthesis of MIP shells by copolymerization of DVB‐80 and methacrylic acid in the presence of TBZ in a mixed solvent porogen (acetonitrile/toluene). The materials were characterized by elemental microanalysis, nitrogen sorption porosimetry and scanning (and transmission) electron microscopy. Thereafter, the imprinted materials were assessed as stationary phases in liquid chromatography. From this study it can be concluded that grafted MIP beads can be obtained in a simple and direct manner, consuming only a fraction of the reagents used typically to prepare imprinted particles from a monolithic imprinted polymer. Such materials can be used in the development of in‐line molecularly imprinted solid‐phase extraction methods. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1058–1066, 2010     

溶菌酶分子印迹聚合物膜的制备及其吸附性能

以溶菌酶为模板蛋白质，结合分子印迹技术在硅烷化的基质玻片上制备了溶菌酶分子印迹聚合物膜。实验优化了溶菌酶聚合物膜的印迹体系，考察了溶菌酶分子印迹聚合物膜的吸附平衡时间、最大吸附量、特异识别能力、重复使用性以及对实际样品中溶菌酶的分离情况。结果表明，在最优条件下，制备的分子印迹聚合物膜对溶菌酶具有特异吸附能力，印迹因子为3.0，吸附平衡时间为5 min，吸附行为符合Langmuir吸附模型，理论最大吸附量为42.5 mg/g，实际样品中的吸附量为30 mg/g。且此印迹聚合物膜在重复使用5次后，最大吸附量仅下降了5%，具有良好的重复使用性。该方法为复杂生物样品中目标蛋白质的分离富集提供了一种快速、高效的手段。     

A phosphotyrosine-imprinted polymer receptor for the recognition of tyrosine phosphorylated peptides

Hyperphosphorylation at tyrosine is commonly observed in tumor proteomes and, hence, specific phosphoproteins or phosphopeptides could serve as markers useful for cancer diagnostics and therapeutics. The analysis of such targets is, however, a challenging task, because of their commonly low abundance and the lack of robust and effective preconcentration techniques. As a robust alternative to the commonly used immunoaffinity techniques that rely on phosphotyrosine(pTyr)-specific antibodies, we have developed an epitope-imprinting strategy that leads to a synthetic pTyr-selective imprinted polymer receptor. The binding site incorporates two monourea ligands placed by preorganization around a pTyr dianion template. The tight binding site displayed good binding affinities for the pTyr template, in the range of that observed for corresponding antibodies, and a clear preference for pTyr over phosphoserine (pSer). In further analogy to the antibodies, the imprinted polymer was capable of capturing short tyrosine phosphorylated peptides in the presence of an excess of their non-phosphorylated counterparts or peptides phosphorylated at serine.     

槲皮素表面分子印迹聚合物的制备及其应用研究

以接枝双键的凹凸棒土(TM)为载体,槲皮素为模板分子,采用表面印迹技术制备对槲皮素具有特异吸附性能的分子印迹聚合物(MIP)。利用光谱法选择实验条件及对化合物表征。采用静态法研究聚合物对槲皮素的结合性能与识别性能。结果表明,该分子印迹材料对槲皮素具有特异的识别特性和优良的亲和性,提高了传统聚合物的结合率。以该印迹聚合物为固相萃取材料,结合高效液相色谱法,对白菜中的槲皮素进行分离富集,方法回收率为84.0%~90.6%,相对标准偏差低于5.6%。     

二茂铁甲酸分子印迹聚合物的制备和识别机理、结合特性

以二茂铁甲酸（FCA）为模板,选用不同的功能单体制备了一系列分子印迹聚合物,用平衡结合实验考察了它们对模板分子的结合性能。 结果表明,以甲基丙烯酸为功能单体制得的印迹聚合物P1对模板分子有很好的选择性,特异性吸附量ΔCP为23.18 μmol/g,印迹因子IF为2.33,竞争性结合实验结果表明,P1可以将模板分子从结构类似物中分离出来。 Scatchard方程研究表明,在研究的浓度范围内聚合物中形成了一类等价的结合位点,其对模板分子的平衡离解常数K=1.94 mmol/L,最大表观结合量Cpmax=92.33 μmol/g。 研究还表明,FCA的羧基是在聚合物的孔穴中产生识别位点的功能基,模板分子上的羧基与MAA的羧基形成双重氢键作用是分子识别的主要作用力。     

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.     

Efficient synthesis of molecularly imprinted polymers with bio‐recognition sites for the selective separation of bovine hemoglobin

We developed a facile approach to the construction of bio‐recognition sites in silica nanoparticles for efficient separation of bovine hemoglobin based on amino‐functionalized silica nanoparticles grafting by 3‐aminopropyltriethoxylsilane providing hydrogen bonds with bovine hemoglobin through surface molecularly imprinting technology. The resulting amino‐functionalized silica surface molecularly imprinted polymers were characterized using scanning electron microscope, transmission electronic microscopy, Fourier transform infrared spectroscopy, X‐ray photoelectron spectroscopy, and thermogravimetric analysis. Results showed that the as‐synthesized imprinted polymers exhibited spherical morphology and favorable thermal stability. The binding adsorption experiments showed that the imprinted polymers can reach equilibrium within 1 h. The Langmuir isotherm and pseudo‐second‐order kinetic model fitted the adsorption data well. Meanwhile, the imprinted polymers possessed a maximum binding capacity up to 90.3 mg/g and highly selectivity for the recognition of bovine hemoglobin. Moreover, such high binding capacity and selectivity retained after eight cycles, indicating the good stability and reusability of the imprinted polymers. Finally, successful application in the selective recognition of bovine hemoglobin from a real bovine blood sample indicated that the imprinted polymers displayed great potentials in efficient purification and separation of target proteins.     

Preparation, characterization and properties studies of quinine-imprinted polymer in the aqueous phase

The uniform-sized spherical molecularly imprinted polymers were successfully prepared through molecular imprinting technology by two-step seed swelling and mini-emulsion polymerization in the aqueous condition using quinine as template molecules and methacrylic acid (MAA) as functional monomer. The polymers were characterized by IR spectra, thermal-weight analysis, scanning electron microscope and laser particle size analysis. The properties of imprinted polymers were investigated in different organic phases and aqueous media. In the organic media, results suggested that polar interactions (hydrogen bonding, ionic interactions) between acidic monomer/polymer and template molecules are mainly responsible for the binding and recognition; whereas in the aqueous medium, a considerable recognition effect was also obtained where the ionic (electrostatic) interaction and hydrophobic interaction play an important role. The experiments of binding different substrates indicated that the MIPs possessed an excellent rebinding ability and inherent selectivity to quinine. __________ Translated from Zhongshan Dcocue Xuebao/Acta Scientianum Natralium University Sunyatseni, 2005, 44(3)(in Chinese)     

分子印迹聚合物微球的制备及应用研究进展

球形分子印迹聚合物具有制备简单、使用方便；分子识别效率高且便于功能设计等优点，近年来成为分子印迹技术领域研究的热点之一。对球形分子印迹聚合物微球的制备及其应用研究进展作了较为详细的介绍。     

利用辅助识别聚合物链制备牛血清白蛋白分子印迹聚合物

论文提出了一种新的蛋白质分子印迹方法, 即以聚乙烯醇接枝聚合物作为辅助识别聚合物链(ARPCs), 以丙烯酰胺为单体, 在丙烯酸酯树脂载体表面进行聚合, 制备牛血清白蛋白分子印迹树脂. 实验使用2.00 mol•L⁻¹氯化钾(KC1)溶液除去模板蛋白质, 使用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测吸附蛋白质, 并设计使用0.150, 0.500和2.00 mol•L⁻¹KC1溶液分别对吸附蛋白质进行梯度洗提. 电泳实验结果表明, 2.00 mol•L⁻¹KC1溶液洗脱的蛋白质是大孔树脂的吸附效应; 0.500 mol•L⁻¹KC1溶液洗脱掉的是印迹蛋白质. 引入ARPCs制得的分子印迹树脂, 用于混合蛋白质体系吸附时, 对模板蛋白质的吸附量为80~100μg•g⁻¹, 其特异性吸附能力较未引入ARPCs之前有明显提高.