蛋白质分子印迹聚合物研究

分子识别现象在生命过程中扮演着极其重要的角色,对此进行研究和探索是化学和生物学领域的核心课题,制备具有分子识别能力的人工受体是对该领域科学工作者的一个持久挑战．为此,相关研究人员进行了多方努力,创造性地发展了许多崭新的方法和手段,其中的分子印迹聚合物制备技术就是这些方法和手段中的一种.     

Chiral recognition and separation of beta2-amino acids using non-covalently molecularly imprinted polymers

Non-covalently molecularly imprinted polymers (MIPs) for beta2-amino acids were prepared for the first time. N-(2-chlorobenzyloxycarbonyl)-(R)-beta2-homophenylalanine (N-2-ClZ-(R)-beta2-HPhe) was imprinted with methacrylic acid (MAA) and/or 4-vinylpyridine (4-VPy) as the functional monomers, with ethylene glycol dimethacrylate (EDMA) as the cross-linker. The MIPs made with different ratios of MAA:4-VPy were studied in HPLC mode. The results show that MIPs made with 4-VPy yielded the best chiral separation factor (alpha= 1.86) for the template molecule. The importance for an efficient separation of pi-stacking interactions between the MIPs and the template molecule is demonstrated. Racemates of Z-alpha-amino acids and beta-amino acid analogues of the template were either not or poorly resolved by the MIPs, thus demonstrating the close three-dimensional complementarity of the MIPs' recognition sites with the template.     

Development of a broad selective molecularly imprinted polymers-based solid phase extraction of contraceptive drug levonorgestrel from water samples

A broad selective molecularly imprinted polymers-based solid phase extraction (MISPE) for levonorgestrel (LNG) from water samples was developed. Using LNG as a template molecule, acrylamide (AA) as functional monomer, ethylene glycol dimethacrylate (EGDMA) as linking agent and bulk polymerisation as a synthetic method, the molecularly imprinted polymers (MIPs) were synthesised and characterised. The MIPs displayed a high specific rebinding for LNG with the imprinting factor of 3.71. The Scatchard analysis showed that there was at least one class of binding site for LNG formed in the MIPs with the dissociation constant of 8.046?µg?mL^?1. The results of selectivity testing indicated that the MIPs also exhibited high cross-reactivity with structurally related compounds (estrone, methylprednisolone and ethinyl estradiol), but no recognition with non-structurally related compound (indomethacin), suggesting that the MIPs could be used as a broad recognition absorbent. MISPE column was prepared by packing MIPs particles into a common SPE cartridge. The MISPE extraction conditions including loading, washing and eluting solutions were carefully optimised. Water samples spiked with LNG were extracted by MISPE column and detected by high-performance liquid chromatography. The recoveries were found to be 79.97?～?132.79% with relative standard deviations (RSD) of 1.92?～?10.43%, indicating the feasibility of the prepared MIPs for LNG extraction.     

Quercetin molecularly imprinted polymers: Preparation, recognition characteristics and properties as sorbent for solid-phase extraction

Molecular imprinted polymers (MIPs) were prepared through thermal polymerization by using quercetin as the template molecule, acrylamide (AA) as the functional monomer and ethylene glycol dimethacrylate (EDMA) as the cross-linker in the porogen of tetrahydrofuran (THF). The synthesized MIPs were identified by both Fourier transform infrared (FTIR) and scanning electron microscope (SEM). Systematic investigations of the influences of key synthetic conditions, including functional monomers, porogens and cross-linkers, on the recognition properties of the MIPs were conducted. Scatchard analysis revealed that the homogeneous binding sites were formed in the polymers. Besides quercetin, two structurally similar compounds of rutin and catechol were employed for molecular recognition specificity tests of MIPs. It was observed that the MIPs exhibited the highest selective rebinding to quercetin. Accordingly, the MIPs were used as a solid-phase extraction (SPE) sorbent for the extraction and enrichment of quercetin in cacumen platycladi samples, followed by HPLC-UV analysis. The application of MIPs with high affinity and excellent stereo-selectivity toward quercetin in SPE might offer a novel method for the enrichment and determination of flavonoid compounds in the natural products.     

L-卡尼丁分子压印聚合物作为手性分离色谱固定相的研究

以L－卡尼丁为模板分子，分别以α－甲基丙烯酸和丙烯酰胺为功能单体，乙二醇二甲基丙烯酸酯为 剂，采用分子压印技术合成了对L－卡尼丁具有高选择性的分子压印聚合物。将所得聚合物用作高效液相色谱固定相，研究了它们对外消旋卡尼丁盐酸盐的拆分能力，分析结果表明，α－甲基丙烯酸作为功能单体所得聚合物对外消旋卡尼丁盐酸盐具有良好的拆分作用，其分离因子α为1．89。     

核壳结构的萘夫西林磁性分子印迹聚合物微球的制备及性能

以表面修饰双键的Fe₃O₄@SiO₂纳米颗粒为基体,以萘夫西林(nafcillin)为模板,甲基丙烯酸(MAA)为单体,乙二醇二甲基丙烯酸酯(EGDMA)为交联剂,偶氮二异丁腈(AIBN)为引发剂,采用三步升温聚合法合成了核壳结构的萘夫西林磁性分子印迹聚合物。采用傅里叶变换红外光谱仪(FT-IR)、透射电子显微镜(TEM)、X射线衍射仪(XRD)和振动样品磁强计(VSM)对制备的印迹聚合物微球进行了表征,得到的磁性印迹聚合物微球的粒径在320 nm左右,大小均匀,分散性较好,可以在外加磁场下与溶剂实现快速分离。对磁性印迹和非印迹聚合物进行了吸附性能研究,结果表明该印迹聚合物微球对模板分子具有很高的吸附容量(50.7 mg/g),特异性识别性能良好(印迹因子为2.46),有望应用于实际样品中萘夫西林残留量的富集分析。     

New configurations and applications of molecularly imprinted polymers

Molecularly imprinted polymers (MIPs) are applicable in a variety of different configurations. For example, bulk polymers imprinted with beta-lactam antibiotics are presented to be used as stationary phases for the chromatographic separation of beta-lactam antibiotics with both aqueous and organic mobile phases. However, in some analytical applications, monosized spherical beads are preferred over the currently used ground bulk polymers. A precipitation polymerization technique allows preparation of monosized spherical imprinted beads with diameters down to 200 nm having excellent recognition properties for different target molecules. Nevertheless, with current imprinting protocols a substantial amount of template has to be used to prepare the polymer. This can be problematic if the template is poorly soluble, expensive or difficult to obtain. It is shown that for analytical applications, the functional monomer:template ratio can be drastically increased without jeopardizing the polymer's recognition properties. Furthermore, a substantial reduction of the degree of crosslinking is demonstrated, resulting in much more flexible polymers that are useful for example the preparation of thin imprinted films and membranes for sensors. Apart from analysis, MIPs also are applicable in chemical or enzymatic synthesis. For example, MIPs using the product of an enzyme reaction as template are utilized for assisting the synthetic reaction by continuously removing the product from the bulk solution by complexation. This results in an equilibrium shift towards product formation.     

替米考星分子印迹聚合物的制备及其固相萃取研究

以泰乐菌素为虚拟模板分子,甲基丙烯酸为功能单体,乙二醇二甲基丙烯酸酯为交联剂,合成了对替米考星具有高选择性的分子印迹聚合物。考察了二甲基甲酰胺、甲醇、丙酮和氯仿4种致孔剂对合成聚合物性能的影响。通过正交实验优化的聚合配方为:1.0mmol泰乐菌素,8.0mmol甲基丙烯酸,20.0mmol乙二醇二甲基丙烯酸酯,6.0mL氯仿,20.0mg偶氮二异丁腈。研究了将该聚合物作为固相萃取填料分离、富集替米考星的萃取条件和萃取性能。当萃取柱依次用甲醇和水(pH9.0)活化,乙腈溶液上样,甲醇和氯仿分别洗涤,3mL氨化甲醇(95:5,V/V)洗脱时,替米考星在分子印迹固相萃取柱上的回收率达到90%以上,而非分子印迹固相萃取柱的回收率仅为32%。     

Synthesis of Molecularly Imprinted Polymer for Removal of Effective Impurity (Benzhydrol) from Diphenhydramine Hydrochloride Drug

The present study describes the synthesis and preliminary testing of molecularly imprinted polymers (MIPs) as scavenger resins for removal of the genotoxic impurities (GTI) benzhydrol from active pharmaceutical ingredients (API). A new molecularly imprinted polymer was synthesized using benzhydrol (template molecule), methacrylic acid (functional monomer), ethylene glycol dimethacrylate (cross‐linker), 2,2′‐azobisisobutironitril (intiator) and chloroform (porogenic solvent). To compare the performance of this polymer, a control polymer or non‐imprinted polymer (NIP) was prepared under the same conditions without the use of template molecule. The synthesized polymers were characterized by FT‐IR spectroscopy. Selectivity of the molecularly imprinted polymer for absorption benzhydrol impurities through adsorption experiments reviews and the results were compared with the adsorption of impurities by NIP. Various parameters were optimized such as time, pH, type of eluent for elution of impurities from polymer, concentration of sample and saturation of polymer. The proposed method was applied for removal of benzhydrol from Diphenhydramine hydrochloride syrup and passing it through the MIPs led to the quantitative removal of benzhydrol.     

软骨藻酸分子印迹聚合物的制备及其固相萃取应用

以贝毒-软骨藻酸的结构类似物1,3,5-戊烷三羧酸为模板分子,4-乙烯基吡啶为功能单体,乙二醇二甲基双丙烯酸酯为交联剂,合成了对软骨藻酸具有较好选择性的分子印迹聚合物。经索氏提取去除模板分子后,在聚合物内部形成了与模板分子1,3,5-戊烷三羧酸以及结构类似物软骨藻酸尺寸、形状以及活性基团互补的结合位点。通过静态平衡结合实验研究了该聚合物的结合能力和选择性能,与化学组成相同的相应非印迹聚合物相比,1,3,5-戊烷三羧酸的分子印迹聚合物对软骨藻酸具有较高的吸附性能和选择性。用150mg印迹聚合物填充于1.0mL玻璃注射器制成的分子印迹固相萃取柱,采用离线模式,并结合高效液相色谱实现紫贻贝和海水中软骨藻酸的分离与检测。对于加标2mg/L的紫贻贝和海水样品,回收率分别达到(93.4±4.9)%和(89.7±3.2)%(n=3)。     

Molecularly imprinted polymers for solid-phase extraction and solid-phase microextraction: recent developments and future trends

Molecularly imprinted polymers (MIPs) are synthetic polymers having a predetermined selectivity for a given analyte, or group of structurally related compounds, that make them ideal materials to be used in separation processes. In this sense, during past years a huge amount of papers have been published dealing with the use of MIPs as sorbents in solid-phase extraction, namely molecularly imprinted solid-phase extraction (MISPE). Although the majority of these papers were restricted to describe the use of different templates for different applications, several attempts proposing new alternatives to minimize the inherent drawbacks of the preparation and use of MIPs (i.e. template bleeding, tedious synthesis procedure, etc.) have been reported. Thus, this paper does not pretend to be a collection of MISPE-related papers but to give an overview on the significant attempts carried out during recent years to improve the performance of MIPs in solid-phase extraction. In addition, the use of MIPs packed in high performance liquid chromatography (HPLC) columns for the direct injection of crude sample extracts and the preparation of imprinted fibres for solid-phase microextraction will be also discussed.     

碳纳米管表面绿原酸印迹固相萃取材料的制备及应用

在多壁碳纳米管表面接枝的双键键合,以绿原酸为模板,甲基丙烯酸为功能单体,乙二醇二甲基丙烯酸酯为交联剂,采用沉淀聚合技术,在碳纳米管表面成功制备绿原酸印迹材料.采用红外光谱、扫描电镜和热重分析研究此印迹材料的性能.结果表明,在碳纳米管表面接枝一层稳定、均匀、30～40 nm厚的印迹材料.采用高效液相色谱研究此印迹材料的吸附动力学及吸附容量,实验结果表明,此印迹材料对绿原酸的结合存在两个结合位点,最大吸附容量Qmax分别为21.5和32.7 μmol/g.以此印迹材料作为固相萃取剂,优化萃取条件,成功应用于金银花提取液中绿原酸的富集分离研究,富集因子达25.     

Thermal preparation of lysozyme-imprinted microspheres by using ionic liquid as a stabilizer

Thermal preparation of lysozyme-imprinted microspheres was firstly investigated by using biocompatible ionic liquid (IL) as a thermal stabilizer. The imprinted microspheres made with IL could obtain the good recognition ability to template protein, whereas the imprinted polymer synthesized in the absence of it had a similar adsorption capacity to the non-imprinted one. Furthermore, the preparation conditions of imprinted polymers (MIPs) including the content of IL, temperature of polymerization, and types of functional monomers and crosslinkers were systematically analyzed via circular dichroism spectrum and activity assay. The results illustrated that using hydroxyethyl acrylate as the functional monomer, ethylene glycol dimethacrylate as the crosslinker, 5 % IL as the stabilizer, and 75 °C as the reaction temperature could retain the structure of template protein as much as possible. The obtained MIPs showed excellent recognition ability to the template protein with the separation factor and selectivity factor value of 4.30 and 2.21, respectively. Consequently, it is an effective way to accurately imprint and separate template protein by cooperatively using circular dichroism spectroscopy and activity assay during the preparation of protein MIPs. The method of utilizing IL to stabilizing protein at high temperature would offer a good opportunity for various technologies to improve the development of macromolecules imprinting. Graphical Abstract

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利多卡因分子印迹聚合物的制备及其在固相萃取中的应用

以利多卡因(LD)分子印迹聚合物为研究体系,利用分子模拟计算功能单体与模板分子之间的相互作用能,并通过作用实验加以验证,确定衣康酸为最佳单体。以乙二醇二甲基丙烯酸酯为交联剂,N,N-二甲基甲酰胺(DMF)为溶剂,采用本体聚合法合成分子印迹聚合物(MIP);采用高效液相色谱检测。实验发现,聚合物具有较高的印迹指数和选择性;在乙腈加载、5%甲醇-乙腈淋洗、5%HCl-甲醇洗脱条件下,聚合物具有最佳印迹效果。通过渗漏实验考察聚合物键合容量。实验发现,在乙腈溶液中每100mg MIP与空白聚合物(NIP)可分别键合1.62和0.92mg LD分子;采用光度法测定牛血清中利多卡因的含量,其平均回收率达到84.6%。     

Highly stereoselective, uniformly sized molecularly imprinted polymers for cinchona alkaloids in hydro-organic mobile phases.

Highly stereoselective, uniformly sized molecularly imprinted polymers (MIPs) for cinchona alkaloids, cinchonine (CN) and cinchonidine (CD), were prepared using methacrylic acid (MAA) as a functional monomer and ethylene glycol dimethacrylate (EDMA) as a cross-linker. The MIPs were evaluated using a mixture of phosphate buffer and acetonitrile as the mobile phase. The CN- and CD-imprinted MAA-co-EDMA polymers can recognize the respective template molecule more than the other diastereomer, and afford an excellent diastereomer separation of CN and CD. In addition, the MIPs gave diastereomer separations of structurally related compounds, quinidine and quinine. The retentive and stereoselective properties of those compounds on the MIPs suggest that electrostatic and hydrophobic interactions can work to recognize these compounds. Furthermore, thermodynamic studies reveal that the entropy-driven effect is significant at mobile-phase pH 5.4, while the enthalpy-driven interactions seem to be dominant at mobile-phase pH 9.6.     

Molecularly imprinted polymer microspheres for solid-phase extraction of protocatechuic acid in Rhizoma homalomenae

Molecularly imprinted polymers (MIPs) had been prepared by precipitation polymerization method using acrylamide as the functional monomer, ethylene glycol dimethacrylate as the cross-linker, acetonitrile as the porogen solvent and protocatechuic acid (PA), one of phenolic acids, as the template molecule. The MIPs were characterized by scanning electron microscopy and Fourier transform infrared, and their performance relative to non-imprinted polymers was assessed by equilibrium binding experiments. Six structurally similar phenolic acids, including p-hydroxybenzoic acid, gallic acid, salicylic acid, syringic acid, vanillic acid, ferulic acid were selected to assess the selectivity and recognition capability of the MIPs. The MIPs were applied to extract PA from the traditional Chinese medicines as a solid-phase extraction sorbent. The resultant cartridge showed that the MIPs have a good extraction performance and were able to selectively extract almost 82% of PA from the extract of Rhizoma homalomenae. Thus, the proposed molecularly imprinted-solid phase extraction-high performance liquid chromatography method can be successfully used to extract and analyse PA in traditional Chinese medicines.     

Molecularly imprinted polymers for 5-fluorouracil release in biological fluids

The aim of this work was to investigate the possibility of employing Molecularly Imprinted Polymers (MIPs) as a controlled release device for 5-fluorouracil (5-FU) in biological fluids, especially gastrointestinal ones, compared to Non Imprinted Polymers (NIPs). MIPs were synthesized using methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as crosslinking agent. The capacity of the polymer to recognize and to bind the template selectively in both organic and aqueous media was evaluated. An in vitro release study was performed both in gastrointestinal and in plasma simulating fluids. The imprinted polymers bound much more 5-Fu than the corresponding non-imprinted ones and showed a controlled/sustained drug release, with MIPs release rate being indeed much more sustained than that obtained from NIPs. These polymers represent a potential valid system for drug delivery and this study indicates that the selective binding characteristic of molecularly imprinted polymers is promising for the preparation of novel controlled release drug dosage form.     

多壁碳纳米管表面过氧化苯甲酰印迹复合材料的制备及固相萃取应用

以过氧化苯甲酰(BOP)为模板分子,甲基丙烯酸(MAA)为功能单体,乙二醇二甲基丙烯酸酯(EGDMA)为交联剂,偶氮二异丁腈(AIBN)为引发剂,采用热聚合法在多壁碳纳米管(MWNTs)表面制备印迹聚合物(MWNTs-MIPs)。采用红外和热重分析等技术对聚合物结构进行表征。采用液相色谱考察该分子印迹聚合物对过氧化苯甲酰的吸附特性。结果表明该印迹聚合物对过氧化苯甲酰表现出特异性吸附,该印迹聚合物对模板分子存在一种结合位点,其最大表观结合量为56.20 µmol/g。该印迹聚合物成功应用于固相萃取富集面粉中微量过氧化苯甲酰,浓度富集因子为526。     

印迹聚合物改性多壁碳纳米管固相萃取熊果酸

以聚乙二醇改性的碳纳米管为基质,熊果酸为模板分子,甲基丙烯酸为功能单体,乙二醇二甲基丙烯酸酯为交联剂,偶氮二异丁腈为引发剂,采用沉淀聚合法成功制备碳纳米管-印迹复合材料.采用红外光谱和扫描电镜研究该复合印迹材料的结构和形貌.结果表明,在碳纳米管表面接枝一层稳定且均匀的印迹材料.采用静态吸附实验研究此印迹材料的吸附性能,...     

Rational design of molecularly imprinted polymer: the choice of cross-linker

The paper describes a rational approach for the selection of cross-linkers during the development of molecularly imprinted polymers (MIPs). As a model system for this research MIPs specific for the drug zidovudine (AZT) were designed and tested. Three cross-linkers trimethylolpropane trimethacrylate (TRIM), ethylene glycol dimethacrylate (EGDMA) and divinylbenzene (DVB) were studied. The analogue of zidovudine (AZT) ester (AZT-ES) was used as a dummy template. The imprinting factors for all of the polymers in the static adsorption experiments were calculated. The data on the AZT adsorption by control polymers (CP), which were prepared with different cross-linkers without a functional monomer, was also analyzed. DVB was found to be more inert towards zidovudine than EGDMA and TRIM, which was confirmed by both molecular modelling and adsorption experiments. It was demonstrated that DVB-based polymers had a higher imprinting factor (I = 1.85) compared with other tested cross-linked polymers. It was suggested that the selection of the cross-linker should be based on the strength of the interaction with the template: the cross-linker which displays lower binding of the template should be preferential because it generates MIPs with lower non-specific binding and a higher imprinting factor, and therefore specificity. Which cross-linker to use for the preparation of any particular MIP can be determined by analysis of the interactions between the cross-linker and template. This could be done either virtually using computational modelling or by template adsorption using a small library of polymers prepared using different cross-linkers.