Bovine serum albumin-imprinted polymer gels prepared by graft copolymerization of acrylamide on chitosan

A graft copolymerization of acrylamide with N,N′-methylenebisacrylamide on chitosan in aqueous medium was utilized in the synthesis of molecularly imprinted polymer (MIP) gels using bovine serum albumin (BSA) as a template. The effect of the amount of initiator and the molecular weight and amount of chitosan on BSA rebinding was investigated. It was found that the resultant MIP gels based on chitosan-g-polyacrylamide (CS-g-PAM) showed significantly higher imprinting efficiency than those only consisting of polyacrylamide (PAM), and also better than those composed of chitosan/PAM semi-interpenetrating polymer network (CS/PAM-s-IPN). The batchwise adsorption of BSA and some non-template proteins showed that the CS-g-PAM based MIP gels demonstrated a quite significant template binding capacity different from the non-imprinted polymer (NIP) gels, and an obvious binding specificity for BSA. The adsorption isotherms of BSA on the MIP gels were determined and well fitted by a Langmuir model with a maximum binding capacity of 39.49 mg/g wet gels. The adsorption kinetics was analyzed by a linear driving force mass-transfer model. The MIP gels also showed much better stability in rebinding of the imprint molecules than those based on CS/PAM-s-IPN after three adsorption–regeneration cycles.     

Similarly sized protein separation of charge‐selective ethylene‐vinyl alcohol copolymer membrane by grafting dimethylaminoethyl methacrylate

Similarly sized protein separation was investigated using a charge‐selective membrane, which prepared by grafting dimethylaminoethyl methacrylate (DMAEMA) onto ethylene vinyl alcohol copolymer (EVAL) membrane. Bovine serum albumin (BSA) and bovine hemoglobin (BHb) was used as model proteins. P(DMAEMA), the weak cationic polyelectrolyte with ionizable tertiary amine groups, contributed to the charge‐selective separation for BSA and BHb. At pH 6.0, the grafted EVAL membrane surface was positively charged and BSA was negatively charged, while BHb was positively charged. The BSA was adsorbed onto the membrane surface due to electrostatic interaction and the BHb passed through the membrane into the permeate. The charge‐selective behavior resulted in the separation of the similarly sized protein. The maximum separation factors of static adsorption separation for model protein and binary mixture were 32.4 and 37.2, respectively. In the dynamic separation process, the maximum separation factor value was 6.2. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46374.     

用于蛋白质BSA识别的温度/pH双敏印迹聚合物

用N-异丙基丙烯酰胺(NIPAM)和壳聚糖为功能单体,牛血清白蛋白（BSA）为模板蛋白,在改性SiO₂表面制备温度/pH双敏蛋白质印迹聚合物。TEM、FTIR和TG等结果证明印迹层已成功接枝在载体表面。系统研究了聚合物的温度/pH双敏性、吸附容量、吸附动力学、特异性、竞争吸附性及重复性。结果表明,印迹聚合物（MIP）的溶胀率和吸附容量受温度和pH影响较大,高温碱性收缩,低温酸性溶胀。在pH 4.6和35℃下,对0.6mg/mL BSA吸附4h时获得较大的吸附容量（83.74mg/g）,印迹因子为2.02。同时MIP也有较好的特异性和竞争吸附性。重复5次后,吸附容量仍能维持在88%,说明重复性良好。这种新型的温度/pH双敏蛋白质分子印迹合成方法简单,在蛋白质的分离和识别方面有较好的应用前景。     

Electrochemical probing of selective haemoglobin binding in hydrogel-based molecularly imprinted polymers

An electrochemical method has been developed for the probing of hydrogel-based molecularly imprinted polymers (HydroMIPs) on the surface of a glassy carbon electrode. HydroMIPs designed for bovine haemoglobin selectivity were electrochemically characterised and their rebinding properties were monitored using cyclic voltammetry. The electrochemical reduction of bovine oxyhaemoglobin (BHb) in solution was observed to occur at −0.460 V vs (Ag/AgCl) in 150 mM phosphate buffer solution (PBS). When the protein was selectively bound to the MIP, the electrochemical reduction of oxyhaemoglobin could be observed at a similar peak potential of −0.480 V vs (Ag/AgCl). When analysing the non-imprinted control polymer (NIP) interfaced at the electrode, which contained no protein, the peak reduction potential corresponded to that observed for dissolved oxygen in solution (−0.65 V vs (Ag/AgCl)). MIP and NIP (in the absence of protein) were interfaced at the electrode and protein allowed to diffuse through the polymers from the bulk solution end to the electrode. It was observed that whereas NIP exhibited a protein response within 10 min of protein exposure, up to 45 min of exposure time was required in the case of the MIP before a protein response could be obtained. Our results suggest that due to the selective nature of the MIP, BHb arrival at the electrode via diffusion is delayed by the MIP due to attractive selective interactions with exposed cavities, but not the NIP which is devoid of selective cavities.     

Rapid and selective separation for mixed proteins with thiol functionalized magnetic nanoparticles

ABSTRACT: Thiol group functionalized silica-coated magnetic nanoparticles (Si-MNPs@SH) were synthesized for rapid and selective magnetic field-based separation of mixed proteins. The highest adsorption efficiencies of binary proteins, bovine serum albumin (BSA; 66 kDa; pI = 4.65) and lysozyme (LYZ; 14.3 kDa; pI = 11) were shown at the pH values corresponding to their own pI in the single-component protein. In the mixed protein, however, the adsorption performance of BSA and LYZ by Si-MNPs@SH was governed not only by pH but also by the molecular weight of each protein in the mixed protein.     

表面带磺酸基团的聚苯乙烯微球的制备及其对蛋白质的吸附

研究了运用分散聚合法,在乙醇/水混合介质中,制备2-丙烯酰胺基-2-甲基丙磺酸(AMPS)与苯乙烯(St)的共聚微球。运用红外、核磁共振、激光光散射和扫描电子显微镜对功能微球进行表征。阐述了该微球对牛血清蛋白(BSA)的吸附量与吸附时间和pH值的关系。结果表明:功能微球对BSA的吸附先随时间的增长而增大,一段时间后达到平衡吸附。当pH值接近BSA等电点(pI=4.7)时,BSA的吸附量达到最大值。     

Adsorption and recognition of protein molecular imprinted calcium alginate/polyacrylamide hydrogel film with good regeneration performance and high toughness

Protein imprinted calcium alginate/polyacrylamide hydrogel film (CA/PAM MIP) with high toughness was prepared using bovine serum albumin (BSA) as template molecule, sodium alginate and acrylamide as functional monomers, N,N′-methylenebisacrylamide (MBAA) as the covalent cross-linker and CaCl₂ as the ionic cross-linker via UV radiation-reduced polymerization. Factors affecting the adsorption capacity and imprinting efficiency of the BSA-imprinted CA/PAM hydrogel films were investigated, such as ratio of polyacrylamide/sodium alginate, film thickness, MBAA concentration and CaCl₂ concentration. Results showed that the CA/PAM MIP exhibited an obvious improvement in terms of adsorption capacity for BSA compared with non-imprinted polymer (NIP). The adsorption capacity of MIP for BSA reached 22.49 mg/g, which was 2.7 times higher than NIP. The regeneration property of the BSA-imprinted CA/PAM hydrogel was distinctly improved and the imprinting efficiency of CA/PAM MIP maintained 77.95% of the initial value after five repetitions. Single and binary proteins rebinding indicated that the CA/PAM MIP exhibited good recognition performance. Cell culture experiments showed CA/PAM MIP was more suitable for cell culture than CA/PAM NIP. The residual sodium dodecyl sulfate (SDS) in the elution process leaded to the death of mouse fibroblast cells (L929) after 3 days. A moderate elution solution without residue eluent should be used to prepare MIP for cell culture.     

荧光光谱法研究茶碱与血红蛋白的相互作用

运用荧光光谱法研究了茶碱与牛血红蛋白(BHb)的相互作用,茶碱对牛血红蛋白的荧光猝灭过程主要为静态猝灭。测定了不同温度下该反应的结合常数、结合位点数及结合热力学参数,热力学参数的变化,表明上述作用过程是一个熵增加、自由能降低的自发分子间作用过程,而且是以疏水作用为主。用同步荧光技术考察了茶碱对BHb构象的影响。     

蛋白质水力学半径的测定及用于蛋白质变性过程的实时监控

采用纳米粒度分析仪Zetasizer Nano测定了牛血清白蛋白（BSA）的水力学半径,考察了pH、离子强度、表面活性剂等的影响,并用于BSA热变性和脲变性过程的实时跟踪。随pH增大,BSA水力学半径呈“U”形变化趋势;酸性条件下,分子膨胀,随着盐浓度升高,BSA分子先小幅减小后显著增大;中性pH范围,离子强度对蛋白质分子尺寸影响很小,蛋白质性质相对稳定。离子型表面活性剂在蛋白质表面吸附,从而影响BSA水力学半径。通过水力学半径的测量实时跟踪蛋白质变性过程的分子变化,发现增加热变性中离子强度可加快变性速率,SDS加入增大了BSA变性温度Tm。脲在促使BSA分子扩张的同时,对链伸展有抑制作用;在DTT存在下,BSA的水力学半径随着时间变化逐渐增大。结果表明,简便的水力学半径测量可以用于蛋白质大小的表征,并可实时跟踪蛋白变性过程的分子尺度变化。     

牛白蛋白包裹复合磁性微球的制备及性能表征

首先在反相(W/O)微乳液体系中制备出Fe3O4/PAM(聚丙烯酰胺)磁性微球;然后在牛白蛋白的磷酸氢二钠-柠檬酸缓冲溶液中,以戊二醛为交联剂,加入Fe3O4/PAM微球,使蛋白质特异性吸附在微球上。采用电子衍射、透射电镜、差热、热重、红外光谱等方法对复合微球的粒径、结构等性质进行了表征与分析。并考察了牛白蛋白的浓度、pH以及保温时间对蛋白质吸附程度的影响。结果显示,微球粒径20 nm左右,粒径均匀;微球具有较大吸附量;pH增大使蛋白质的吸附量减小;在一定范围内,增大蛋白质初始浓度和延长保温时间均有利于增加蛋白质的吸附量。     

红霉素分子印迹聚合物纳米微球的制备及其吸附特性

以红霉素为模板分子、甲基丙烯酸为功能单体、乙二醇二甲基丙烯酸酯为交联剂,采用沉淀聚合法制备了粒径均一的红霉素纳米分子印迹聚合物微球,优化了分子印迹聚合物的合成条件,确定了模板分子与功能单体的最佳摩尔比为1:3,对其进行了表征. 结果表明,所制聚合物对红霉素的实际最大吸附量可达202.12 mg/g,吸附约200 min达到平衡,对红霉素具有良好的选择性吸附能力.     

复相乳化法制备海藻酸钙微球及其释放行为

采用复相乳化法制备了载牛血清白蛋白(BSA)的海藻酸钙微球,通过正交实验和单因素分析,以BSA包埋率、微球的载药率和平均粒径为考察指标,优化了该方法的制备参数,使最终制备的微球平均粒径小于10 mm,球形度较好,包埋率约70%,载药率达4%. 随着海藻酸钠质量分数的降低和BSA质量的增大,微球的包埋率下降、载药率升高、平均粒径减小. 微囊化BSA的体外释放曲线表明,该系统存在pH响应特性,尤其在磷酸缓冲液中,被包埋BSA的释放速率较快. 电泳结果表明,BSA的分子结构并未受制备过程的影响. 因此,该微囊化方法有望用于蛋白类药物的控释制剂,使其免受胃酸等的破坏,达到肠部释药的目的.     

Preparation and characterization of magnetic poly(styrene–glycidyl methacrylate) microspheres for highly efficient protein adsorption by two‐stage dispersion polymerization

Micrometer‐sized superparamagnetic poly(styrene–glycidyl methacrylate)/Fe₃O₄ spheres were synthesized by two‐stage dispersion polymerization with modified hydrophobic Fe₃O₄ nanoparticles, styrene (St), and glycidyl methacrylate (GMA). The morphology and properties of the magnetic Fe₃O₄–P (St‐GMA) microspheres were examined by scanning electron microscopy, transmission electron microscopy, vibrating sample magnetometry, thermogravimetric analysis, and attenuated total reflectance. The average size of the obtained magnetic microspheres was 1.50 μm in diameter with a narrow size distribution, and the saturation magnetization of the magnetic microspheres was 8.23 emu/g. The magnetic Fe₃O₄–P (St‐GMA) microspheres with immobilized iminodiacetic acid–Cu²⁺ groups were used to investigate the adsorption capacity and selectivity of the model proteins, bovine hemoglobin (BHb) and bovine serum albumin (BSA). We found that the adsorption capacity of BHb was as high as 190.66 mg/g of microspheres, which was 3.20 times greater than that of BSA, which was only 59.64 mg/g of microspheres as determined by high‐performance liquid chromatography. With a rather low nonspecific adsorption, these microspheres have great potential for protein separation and purification applications. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43005.     

Development of a molecularly imprinted polymer for the recovery of lactoferrin

Lactoferrin (LF) was recovered by molecular imprinting using either vinylpirydin alone (MIP1) or mixed with methacrylic acid (MIP2) as functional monomers. In order to create the specific cavity, pure LF was used as template. Controls were prepared for every MIP. The polymers obtained were tested against a protein mixture containing LF, and the recovery efficiency was calculated determining the diminution of LF from the supernatant by titration of the iron of the LF, as well as native PAGE to determine the protein composition of the supernatants. The only polymer which was able to specifically bind LF was MIP1 (vinylpirydin alone). Measuring the proportions of the different proteins in the mixture, it was demonstrated that before the separation with MIP1, LF represented 81.9% of the total protein content of the mixture, and diminished to 70.5% after the exposure to MIP1, suggesting that LF was specifically sorbed by this polymer. The retention efficiency of the polymer showed that MIP1 retained 34.5% of the total LF content, while its control did not retain any, demonstrating that the retention of the protein is not due to unspecific adsorption in the polymer, but rather to a selective retention in the cavity formed by the template.     

混合模式吸附中辛酸钠和牛血清白蛋白间相互作用

混合模式吸附是一种抗体分离新方法,适量添加辛酸钠,可以减弱血清白蛋白等杂质吸附,提高抗体结合的选择性。本文以MEP HyperCel介质和牛血清白蛋白（BSA）为模型,结合静态吸附平衡和等温滴定量热（ITC）法,考察了辛酸钠浓度、pH、盐和温度等影响,探讨了辛酸钠的作用机制。随辛酸钠浓度增大,BSA和牛血免疫球蛋白的饱和吸附量均呈现先降低后升高的趋势,不过辛酸钠对BSA影响更显著,ITC分析表明辛酸钠与BSA之间存在较强的相互作用,且以疏水作用为主导。添加辛酸钠后,BSA吸附量随pH或温度升高而降低,不同盐具有不同效果,ITC分析表明pH、盐和温度均不同程度影响辛酸钠-BSA相互作用,从而影响MEP HyperCel吸附BSA。结果表明,等温滴定量热分析与宏观吸附现象相一致,可量化分析小分子-蛋白间相互作用,为研究蛋白吸附过程中小分子添加物的作用机制提供了新思路。     

pH值和离子强度对空间质量作用模型参数的影响

以牛血清白蛋白（BSA）为模型蛋白质研究了pH值和离子强度对空间质量作用模型参数的影响。通过静态吸附实验确定了不同pH值和离子强度条件下BSA在阴离子交换剂DEAE-Spherodex M上的吸附平衡,利用色谱实验及非线性最小二乘法确定模型参数。研究发现,只有在pH值远大于BSA等电点时,蛋白质的特征电荷数和空间因子才为常数;离子交换平衡常数不仅是pH值的函数,也受到液相离子强度的影响。     

高效制备液相色谱法纯化生物制品的实验研究

以牛血清白蛋白和牛血清红蛋白混合物的分离和工业级枯草杆菌中性蛋白酶的纯化为实验物系，在不同的操作条件下，对自行研制的高效制备液相色谱装置的性能进行了实验研究。结果表明，在适宜的操作条件下，此装置的分离和纯化效果令人满意。     

水溶性壳聚糖纳米粒子的制备及其BSA载药性能

为了避免高分子量壳聚糖水溶性差以及增溶剂乙酸可能带来的负面作用,本文选择低分子量水溶性壳聚糖 (WSC)作研究对象,采用三聚磷酸(TPP)作交链剂制备不同WSC/TPP比率的WSC纳米粒子,并用于牛血清白蛋白 (BSA)的释放载体。经测得为球形形貌的纳米粒子空载和载药时粒径、Zeta电位分别在35～190 nm、35～42 mV。红外光谱及X–射线衍射证实了纳米粒子中WSC的氨基与TPP的磷酸基团发生了交联反应。纳米粒子载药性能试验表明在0.05～1 mg/mL范围内随着BSA浓度的增大,纳米粒子的载药量增加而负载率降低。体外释放实验表明水溶性壳聚糖纳米载体对蛋白质药物具有缓释特征。因此,水溶性壳聚糖有望成为新的载体应用于蛋白质药物的控制释放。     

壳聚糖修饰磁性纳米粒子对BSA的吸附性能

采用化学共沉淀法制备磁性Fe3O4纳米粒子,以（3-氯丙基）三甲氧基硅烷为偶联剂将壳聚糖共价键合到磁性Fe3O4纳米粒子的表面,通过红外光谱（FTIR）、X射线衍射（XRD）、扫描电子显微镜（SEM）及热重分析（TGA）对其进行了表征。主要研究了不同影响因素（吸附时间、pH值、牛血清白蛋白浓度）下壳聚糖修饰的磁性纳米粒子对牛血清白蛋白（BSA）的吸附性能。结果得到壳聚糖修饰的磁性Fe3O4纳米粒子粒径为20 nm左右,壳聚糖在磁性Fe3O4纳米粒子表面的接枝率为15.40%。研究表明：在不同条件下,与未修饰的磁性Fe3O4纳米粒子相比,经壳聚糖修饰的Fe3O4纳米粒子对BSA均表现出较强的吸附能力。     

Acrylonitrile-based copolymer membranes containing reactive groups: effects of surface-immobilized poly(ethylene glycol)s on anti-fouling properties and blood compatibility

The anti-fouling properties and blood compatibility of poly(acrylonitrile-co-maleic acid) (PANCMA) membranes were improved by the immobilization of poly(ethylene glycol)s (PEG) on membrane surface. It was found that the reactive carboxyl groups on PANCMA membrane surface could be conveniently conversed into anhydride groups and then esterified with PEG. Chemical and morphological changes as well as biocompatibility on membrane surface were analyzed by Fourier transform infrared spectroscopy, elemental analysis, scanning electron microscopy, water contact angle, protein adsorption, and platelet adhesion. Results revealed that, with the immobilization of PEG, the hydrophilicity and blood compatibility of the acrylonitrile-based copolymer membranes were improved obviously. The molecular weight of PEG had an obvious influence on the properties of the PEG-immobilized membranes. Permeation behaviors for the studied membranes were investigated by water and bovine serum albumin (BSA) filtration experiments. Compared with the original PANCMA membrane, the membrane immobilized with PEG 400 (M_w=400 g/mol) showed a three-fold increase in a BSA solution flux, a 40.4% reduction in total fouling, and a 57.9% decrease in BSA adsorption.