1-乙烯基-3-甲基咪唑离子液体/分子印迹材料用于牛血红蛋白的识别

分子印迹聚合物是一类对目标分子具备特异性辨别能力的高分子吸附剂材料.运用本体聚合法,以牛血红蛋白(BHb)为模板分子,丙烯酰胺(AAM)和碘化1-乙烯基-3-甲基咪唑离子液体在交联剂、引发剂和加速剂的作用下进行聚合,制备的分子印迹材料对牛血红蛋白(BHb)具备特异性识别功能.同时,对识别条件进行了优化和讨论.     

壳聚糖包裹硅胶载体印迹牛血红蛋白的研究

用壳聚糖包裹的硅胶为载体, 利用壳聚糖表面的氨基与戊二醛结合, 在硅胶表面引入醛基, 固定模板蛋白(牛血红蛋白). 用溶胶-凝胶过程再次包裹固定蛋白质的载体, 洗去模板蛋白后, 得到具有选择性识别的牛血红蛋白分子印迹聚合物. 用红外光谱(IR)、扫描电子显微镜(SEM)和元素分析对聚合物进行了表征, 结果表明, 载体表面成功地接枝了分子印迹聚合物. 选择性吸附实验结果表明, 分子印迹聚合物具有良好的识别性能, 能实现水溶液中牛血红蛋白的富集.     

Investigation of protein imprinting in hydrogel-based molecularly imprinted polymers (HydroMIPs)

We have developed a strategy to produce molecularly imprinted polymers based on polyacrylamide hydrogels for the selective imprinting of bovine haemoglobin (BHb). For the first time, we have explored in detail a variety of template removal strategies including varying ratios of sodium dodecylsulphate:acetic acid (SDS:AcOH) and also the use of a trypsin digest. The optimum ratio of SDS:AcOH was found to be a 10% (w/v):10% (v/v) for the most effective template removal. This resulted in >90% (imprinting efficiency) of re-loaded template (protein) molecule being selectively bound within the MIP. At 15%:15% of SDS:AcOH, although there was even more initial template removal, subsequent re-binding studies showed a decrease in imprinting efficiency (67.9%). Trypsin solutions were also used as a method of template removal. Up to 87.4% of template was reproducibly removed initially; however, the imprinting efficiency was only 20.4%. The high selectivity of the BHb HydroMIP to BHb compared with other structural analogues (namely cytochrome C and myoglobin) was successfully demonstrated.     

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.     

BSA‐imprinted synthetic receptor for reversible template recognition

A novel approach to the manufacturing of protein‐responsive imprints on a home‐made chitosan substrate was established together with m‐aminophenylboronic acid (APBA) as a functional monomer. The produced polymers were characterized using both (1) equilibrium adsorption assays and (2) high performance liquid chromatography analysis. Results confirmed that the synthesized BSA‐MIP (molecularly imprinted polymer) has a high affinity towards its template compared to the determined control proteins. The produced BSA‐MIP featured largely in its good adsorption reversibility, especially in competitive binding assays, which is of great biological significance in separations. Non‐specific binding was reduced to almost zero in a BSA/BHb competitive binding event. An excellent HPLC profile of template recognition was found for BSA‐MIP, even under harsh mobile phase conditions. In the present work, the adopted trapped‐template‐release method permits recovery of bound BSA [1]. The strategy of making an artificial protein‐receptor with high adsorption affinity and reversibility is promising in on‐line isolation of target protein from complicated biological environments.     

复合分子印迹聚合物体系选择性富集蛋白质样品中的溶菌酶

考察了功能单体与模板蛋白的反应摩尔比、溶液pH值及离子强度对功能单体与模板蛋白之间相互作用的影响, 得出制备分子印迹聚合物的最佳条件. 在最佳条件下, 以溶菌酶(Lyz)为模板分子, 丙烯酰胺(AA)和N,N’-亚甲基双丙烯酰胺(BisAA)为聚合基质, 二氧化硅为固体制孔剂, 制备了复合分子印迹聚丙烯酰胺凝胶, 并用平衡吸附实验研究了其吸附性能和识别选择性. 研究结果表明, 该聚合物对模板蛋白有较高的亲和性、选择性和吸附容量,可以从蛋白质混合溶液中分离富集模板分子.     

Preparation and application of hollow molecularly imprinted polymers with a super‐high selectivity to the template protein

Protein‐imprinted polymers with hollow cores that have a super‐high imprinting factor were prepared by etching the core of the surface‐imprinted polymers that used silica particles as the support. Lysozyme as template was modified onto the surface of silica particles by a covalent method, and after polymerization and the removal of template molecules, channels through the polymer layer were formed, which allowed a single‐protein molecule to come into the hollow core and attach to the binding sites inside the polymer layer. The adsorption experiments demonstrated that the hollow imprinted polymers had an extremely high binding capacity and selectivity, and thus a super‐high imprinting factor was obtained. The as‐prepared imprinted polymers were used to separate the template lysozyme from egg white successfully, indicating its high selectivity and potential application in the field of separation of protein from real samples.     

修饰硅胶表面印迹牛血红蛋白及其识别性能的研究

本文选用马来酸酐修饰后的硅胶作为载体,丙烯酰胺为功能单体,N,N’-亚甲基双丙烯酰胺为交联剂,牛血红蛋白为模板分子,采用氧化还原悬浮聚合法,合成了具有选择性识别的牛血红蛋白分子印迹聚合物。并用红外光谱(IR)、扫描电子显微镜(SEM)对聚合物进行了表征,结果表明载体表面成功接枝了分子印迹聚合物薄层。同时,选择性吸附实验表明分子印迹聚合物的具有良好的识别性能,能成功的实现水溶液中牛血红蛋白的富集。     

Synthesis of magnetic dual‐template molecularly imprinted nanoparticles for the specific removal of two high‐abundance proteins simultaneously in blood plasma

Novel core–shell dual‐template molecularly imprinted superparamagnetic nanoparticles were synthesized using bovine hemoglobin and bovine serum albumin as the templates for the efficient depletion of these two high‐abundance proteins from blood plasma for the first time. The preparation process combined surface imprinting technique and a two‐step immobilized template strategy. The obtained polymers were fully characterized by transmission electron microscopy, X‐ray diffraction, Fourier transform infrared spectroscopy, and vibrating sample magnetometry. The results showed that the as‐synthesized nanomaterials possessed homogeneous and thin imprinted shells with a thickness of about 5 nm, stable crystalline phase, and superparamagnetism. The binding performance of the imprinted polymers was investigated through a series of adsorption experiments, which indicated that the products had satisfactory recognition ability for bovine hemoglobin and bovine serum albumin. The resultant nanoparticles were also successfully applied to simultaneously selective removal of two proteins from a real bovine blood sample.     

Molecularly imprinted polymer grafted on polysaccharide microsphere surface by the sol-gel process for protein recognition

An interfacial organic–inorganic hybridization concept was applied to the preparation of a new spherical imprinted material for protein recognition. The functional biopolymer chitosan (CS), shaped as microsphere and high-density cross-linked, constituted of the polysaccharide core for surface imprinting. After the model template protein, bovine serum albumin, was covalently immobilized by forming imine bonds with the functional amine groups of CS, two kinds of organic siloxane (3-aminopropyltrimethoxysiloxane: APTMS, and tetraethoxysiloxane: TEOS) assembled and polymerized on the polysaccharide–protein surface via sol–gel process in aqueous solution at room temperature. After template removal, the protein-imprinted sol–gel surface exhibited a prevalent preference for the template protein in adsorption experiments, as compared with four contrastive proteins. Bioinformatics methods were also employed to investigate the imprinting process and the recognition effect. The influence of siloxane type, pH, siloxane/water ratio on template removal and recognition selectivity was assessed. Under optimized imprinting conditions, a large quantity of well-distributed pores was observed on the immobilized-template imprinted surface. The surface-imprinted adsorbent offered a fast kinetics for template re-adsorption and could be reused. Compared with the imprinted material prepared with free-template, material prepared with immobilized-template possessed higher adsorption capacity towards template protein. Easy preparation of the described imprinted material, high affinity and good reusability make this approach attractive and broadly applicable in biotechnology for down-stream processing and biosensor.     

Bovine serum albumin surface imprinted polymer fabricated by surface grafting copolymerization on zinc oxide rods and its application for protein recognition

A novel bovine serum albumin (BSA) surface imprinted polymer based on ZnO rods was synthesized by surface grafting copolymerization. It exhibited an excellent recognition performance to bovine serum albumin. The adsorption capacity and imprinting factor of bovine serum albumin could reach 89.27 mg/g and 2.35, respectively. Furthermore, the fluorescence property of ZnO was used for tracing the process of protein imprinting and it implied the excellent optical sensing property of this material. More importantly, the hypothesis that the surface charge of carrier could affect the imprinting process was confirmed. That is, ZnO with positive surface charge could not only improve the recognition specificity of binding sites to template proteins (pI < 7), but also deteriorate the bindings between sites and non‐template proteins (pI > 7). It was also important that the reusability of ZnO@BSA molecularly imprinted polymers was satisfactory. This implied that the poor mechanical/chemical stability of traditional zinc oxide sensors could be solved by the introduction of surface grafting copolymerization. These results revealed that the ZnO@BSA molecularly imprinted polymers are a promising optical/electrochemical sensor element.     

Preparation of ellagic acid molecularly imprinted polymeric microspheres based on distillation–precipitation polymerization for the efficient purification of a crude extract

Molecularly imprinted polymeric microspheres with a high recognition ability toward the template molecule, ellagic acid, were synthesized based on distillation–precipitation polymerization. The as‐obtained polymers were characterized by scanning electron microscopy, infrared spectroscopy, and thermogravimetric analysis. Static, dynamic, and selective binding tests were adopted to study the binding properties and the molecular recognition ability of the prepared polymers for ellagic acid. The results indicated that the maximum static adsorption capacity of the prepared polymers toward ellagic acid was 37.07 mg/g and the adsorption equilibrium time was about 100 min when the concentration of ellagic acid was 40 mg/mL. Molecularly imprinted polymeric microspheres were also highly selective toward ellagic acid compared with its analogue quercetin. It was found that the content of ellagic acid in the pomegranate peel extract was enhanced from 23 to 86% after such molecularly imprinted solid‐phase extraction process. This work provides an efficient way for effective separation and enrichment of ellagic acid from complex matrix, which is especially valuable in industrial production.     

Molecularly imprinted polymer functionalized magnetic Fe3O4 for the highly selective extraction of triclosan

We present a facile strategy to prepare the molecularly imprinted polymers layer on the surface of Fe₃O₄ nanoparticles with core‐shell structure via sol–gel condensation for recognition and enrichment of triclosan. The Fe₃O₄ nanoparticles were first synthesized by a solvothermal method. Then, template triclosan was self‐assembled with the functional monomer 3‐aminopropyltriethoxysilane on the silica‐coated Fe₃O₄ nanoparticles in the presence of ethanol and water. Finally, the molecularly imprinted polymers were formed on the surface of silica‐coated Fe₃O₄ nanoparticles to obtain the product. The morphology, magnetic susceptibility, adsorption, and recognition property of magnetic molecularly imprinted polymers were characterized using transmission electron microscopy, Fourier transform infrared spectroscopy, X‐ray diffractometry, vibrating sample magnetometry, and re‐binding experiments. The magnetic molecularly imprinted polymers showed binding sites with good accessibility, fast adsorption rate, and high adsorption capacity (218.34 μg/g) to triclosan. The selectivity of magnetic molecularly imprinted polymers was evaluated by the rebinding capability of triclosan and two other structural analogues (phenol and p‐chlorophenol) in a mixed solution and good selectivity with an imprinting factor of 2.46 was obtained. The application of triclosan removal in environmental samples was demonstrated.     

Magnetic deep eutectic solvents molecularly imprinted polymers for the selective recognition and separation of protein

A novel and facile magnetic deep eutectic solvents (DES) molecularly imprinted polymers (MIPs) for the selective recognition and separation of Bovine hemoglobin (BHb) was prepared. The new-type DES was adopted as the functional monomer which would bring molecular imprinted technology to a new direction. The amounts of DES were optimized. The obtained magnetic DES-MIPs were characterized with fourier transform infrared spectrometry (FT-IR), thermogravimetric analysis (TGA), field emission scanning electron microscope (FESEM), dynamic light scattering (DLS), elemental analysis and vibrating sample magnetometer (VSM). The results suggested that the imprinted polymers were successfully formed and possessed a charming magnetism. The maximum adsorption capability (Q_max) and dissociation constant (K_L) were analyzed by Langmuir isotherms (R² = 0.9983) and the value were estimated to be 175.44 mg/g and 0.035 mg/mL for the imprinted particles. And the imprinted particles showed a high imprinting factor of 4.77. In addition, the magnetic DES-MIPs presented outstanding recognition specificity and selectivity so that it can be utilized to separate template protein from the mixture of proteins and real samples. Last but not least, the combination of deep eutectic solvents and molecular imprinted technology in this paper provides a new perspective for the recognition and separation of proteins.     

One‐step preparation of magnetic imprinted nanoparticles adopting dopamine‐cupric ion as a co‐monomer for the specific recognition of bovine hemoglobin

A novel magnetic core–shell polydopamine–cupric ion complex imprinted polymer was prepared in one‐step through surface imprinting technology, which could specifically recognize bovine hemoglobin from the real blood samples. The polymerization conditions and adsorption performance of the resultant nanomaterials were investigated in detail. The results showed that the cupric ion played an important role in the recognition of template proteins. The saturating adsorption capacity of this kind of imprinted polymers was 2.23 times greater than those of imprinted polymers without cupric ion. The imprinting factor of the imprinted materials was as high as 4.23 for the template molecule. The selective separation bovine hemoglobin from the real blood sample is successfully applied. In addition, the prepared materials had excellent stability and no obvious deterioration after five adsorption–regeneration cycles. Easy preparation, rapid separation, high binding capacity and satisfactory selectivity for the template protein make this polymer attractive in the separation of high‐abundance proteins.     

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.     

Magnetic Silica‐Coated Sub‐Microspheres with Immobilized Metal Ions for the Selective Removal of Bovine Hemoglobin from Bovine Blood

Magnetic silica‐coated magnetite (Fe₃O₄) sub‐microspheres with immobilized metal‐affinity ligands are prepared for protein adsorption. First, magnetite sub‐microspheres were synthesized by a hydrothermal method. Then silica was coated on the surface of Fe₃O₄ particles using a sol–gel method to obtain magnetic silica sub‐microspheres with core‐shell morphology. Next, the trichloro(4‐chloromethylphenyl) silane was immobilized on them, reacted with iminodiacetic acid (IDA), and charged with Cu²⁺. The obtained magnetic silica sub‐microspheres with immobilized Cu²⁺ were applied for the absorption of bovine hemoglobin (BHb) and the removal of BHb from bovine blood. The size, morphology, and magnetic properties of the resulting magnetic micro(nano) spheres were investigated by using scanning microscopy (SEM), transmission electron microscopy (TEM), X‐ray diffraction (XRD), and a vibrating sample magnetometer (VSM). The measurements showed that the magnetic sub‐microspheres are spherical in shape, very uniform in size with a core‐shell, and are almost superparamagnetic. The saturation magnetization of silica‐coated magnetite (Fe₃O₄) sub‐microspheres reached about 33 emu g^?1. Protein adsorption results showed that the sub‐microspheres had a high adsorption capacity for BHb (418.6 mg g^?1), low nonspecific adsorption, and good removal of BHb from bovine blood. This opens a novel route for future applications in removing abundant proteins in proteomic analysis.     

Synthesis of stationary phases that provide group recognition for polychlorinated biphenyls by porogenic fragment template imprinting

Molecular recognition based on imprinted polymers results from the polymerization of functional monomers and cross‐linkers in the presence of a target analyte (i.e. template), with subsequent removal of the template to create synthetic binding sites. However, complete removal of the template is difficult to achieve, thereby leading to template leaching, which adversely affects real‐world analytical applications. To overcome this challenge, the present study utilizes porogenic fragment template imprinting techniques to provide an alternative synthetic strategy to generate molecularly imprinted polymers with molecular recognition toward polychlorinated biphenyls. Thereafter, thus‐generated imprinted polymers have been applied as stationary phases in molecularly imprinted solid‐phase extraction for preconcentrating six “indicator polychlorinated biphenyls” in both organic and aqueous media. Recoveries of up to 98.9% (imprinted polymers) versus 73.0% (conventional C₁₈) in an organic phase, and up to 97.4% (imprinted polymers) versus 89.4% (C₁₈) in an aqueous phase have been achieved corroborating the utility of this advanced sorbent material. Finally, porogenic fragment template imprinting strategies have yielded molecularly imprinted polymers that are useful for the quantitative determination of polychlorinated biphenyls in environmental matrices, which provides a low‐cost strategy for tailoring stationary phases that avoid template leaching in applications in solid‐phase extraction as well as liquid chromatography.     

Synthesis of thermo‐responsive bovine hemoglobin imprinted nanoparticles by combining ionic liquid immobilization with aqueous precipitation polymerization

Surface molecular imprinting over functionalized nanoparticles has proved to be an effective approach for construction of artificial nanomaterials for protein recognition. Herein, we report a strategy for synthesis of core–shell protein‐imprinted nanoparticles by the functionalization of nano‐cores with ionic liquids followed by aqueous precipitation polymerization to build thermo‐responsive imprinted polymer nano‐shells. The immobilized ionic liquids can form multiple interactions with the protein template. The polymerization process can produce thermo‐reversible physical crosslinks, which are advantageous to enhancing imprinting and facilitating template removal. With bovine hemoglobin as a model template, the imprinted nanoparticles showed temperature‐sensitivity in both dispersion behaviors and rebinding capacities. Compared with the ionic‐liquid‐modified core nanoparticles, the imprinted particles exhibited greatly increased selectivity and two orders of magnitude higher binding affinity for the template protein. The imprinted nanoparticles achieved relatively high imprinting factor up to 5.0 and specific rebinding capacity of 67.7 mg/g, respectively. These nanoparticles also demonstrated rapid rebinding kinetics and good reproducibility after five cycles of adsorption–regeneration. Therefore, the presented approach may be viable for the fabrication of high‐performance protein‐imprinted nanoparticles with temperature sensitivity.     

Preparation,characterization, and binding profile of molecularly imprinted hydrogels for the peptide hepcidin

Molecularly imprinted hydrogels for the capture of the peptide hormone hepcidin were prepared by water‐in‐oil (w/o) suspension polymerization under mild conditions. Spherical and relatively uniformly sized gel beads were routinely obtained after optimization of the synthetic methodology. The polymers were analyzed by Fourier transform infrared spectroscopy, optical microscopy, and scanning electron microscopy. Although the imprinted materials exhibited higher affinity towards the epitope template (hepcidin N‐terminus) than their corresponding blank polymers, the full‐length target peptide was found strongly bound to all the hydrogels tested. However, by using whole fluorescent hepcidin as the print species, the imprinting effect was more pronounced. Moreover, bovine serum albumin did not bind to the poly N‐isopropylacrylamide (PNIPAm)‐based polymers. Thus, polymeric “sponges” for biomacromolecules with size‐exclusion effect were developed, useful for peptide concentration, immobilization and/or purification from serum samples. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1721–1731, 2010