CN106970215A - 一种检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备方法 - Google Patents
一种检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备方法 Download PDFInfo
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- CN106970215A CN106970215A CN201710185685.5A CN201710185685A CN106970215A CN 106970215 A CN106970215 A CN 106970215A CN 201710185685 A CN201710185685 A CN 201710185685A CN 106970215 A CN106970215 A CN 106970215A
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- thifensulfuronmethyl
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Abstract
一种检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备方法,包括在Fe3O4磁性纳米粒子的表面修饰聚乙二醇2000,表面包覆SiO2壳层,形成芯‑壳‑壳结构,通过洗脱位于SiO2壳层中的印记分子,形成具有与印记分子结构、大小和功能基互补的特异性识别位点空穴,实现对目标分析物分子选择性识别和检测。所述的人工抗体制备过程包括如下三个步骤:首先,制得Fe3O4磁性纳米粒子并在其表面修饰聚乙二醇;然后,加入目标分子噻吩磺隆、交联剂和催化剂,水解得表面印记噻吩磺隆的Fe3O4@PEG@SiO2粒子;最后,用体积比为1:4的乙酸和丙酮混合液洗脱模板分子,得到具有选择性识别印记分子的Fe3O4@PEG@SiO2人工抗体,其对噻吩磺隆的最大饱和结合量为41.28mg/g,前30min内,其吸附速率为0.45mg/g·min,分别是非印记方法的5.34倍和3.46倍。
Description
技术领域
本发明涉及材料科学领域,特别涉及人工抗体的制备及其表面印记对噻吩磺隆检测方法。
背景技术
噻吩磺隆(thifensulfuron methyl)是一种磺酰脲类除草剂,因具有较高的除草性能而被广泛用于田间杂草的去除工作。但其在农产品和环境中的残留会给人类健康带来危害,同时造成环境污染。因此,如何快速、准确、有选择性的检测残留在农产品和环境中的噻吩磺隆,是一项有挑战性的难题,也是目前迫切需要解决的问题。
分子印记技术(molecular imprinting technique,MIT)是一种利用分子印记聚合物(molecular imprinting polymers,MIPs)模拟酶-底物或抗体-抗原之间的相互作用,对印记分子(imprinting molecular)也称模板分子(template molecular)进行专一识别的技术。近年来随着高分子化学、材料科学、化学工程及生物化学等交叉学科快速发展,推进了分子印记技术的迭代,主要表现在人工合成分子印记材料(人工抗体)方面,其与天然抗体相比,具有更高的构效预定性、特异识别性和广泛实用性等主要优点。
运用纳米合成技术和分子印记技术制备的功能性的Fe3O4芯-壳结构已经被许多科研团队广泛的开展研究。2015年,杨鑫等人公开了发明专利(CN105254827A)“一种分离烟碱农药哌虫啶的磁性纳米分子印记聚合物”的制备方法,该发明利用三氯化铁、乙二醇、无水乙酸钠放置在高温反应釜中,在温度为180 ~ 220°C条件下反应l h ~ 13 h,停止反应静置15~25min,制得Fe3O4磁性纳米粒子,然后,用盐酸和柠檬酸三钠对其表面进行改性,加入正硅酸乙酯和氨水水解后合成了Fe3O4@SiO2,然后再用硅烷化试剂3-(三甲氧基甲硅烷基)丙基丙烯酸酯水解、用无水甲苯、乙醇洗涤得到Fe3O4@SiO2@C=C复合物颗粒,最后加入目标分子哌虫啶、交联剂乙二醇二甲基丙烯酸酯和引发剂偶氮二异丁晴聚合,得到印记哌虫啶的Fe3O4@SiO2@C=C分子印记聚合物,该发明对哌虫啶的最大饱和吸附量为17.305±0.403mg/g,是非分子印记最大饱和吸附量的2.0倍。该方法过程繁琐,饱和结合量小,吸附动力学慢,高分子聚合物刚性弱,易坍塌,导致有效识别位点数量减少,制备过程用甲苯溶剂洗涤,对环境不友好。2016年,王永强等人公开了发明专利(CN105832699A)“一种Fe3O4@SiO2蛋黄-蛋壳结构中空复合微球的制备方法及应用”。该发明利用FeCl3·6H2O、尿素、柠檬酸三钠的水溶液在加入聚丙烯酸钠的条件下加热制得Fe3O4纳米微球。该方法在后续步骤中以正硅酸乙酯(Tetraethyl orthosilicate,TEOS)作为硅源,经浓盐酸腐蚀后还原得到Fe3O4@SiO2芯-壳结构微球,用于药物的缓释释放。由于浓盐酸的腐蚀性与环境危害性,该方法在实际推广过程中将受到一定限制。2016年,王娜等人公开了发明专利(CN106118630A)“磁光双功能CNT/Fe3O4@SiO2(FITC)一维纳米复合材料的制备方法”。该方法将多壁碳纳米管、乙酰丙酮铁、三甘醇加热1小时后制得CNT/Fe3O4,随后利用异硫氰酸荧光素(Fluoresceinisothiocyanate,FITC)与3-氨丙基三乙氧基硅烷在乙醇中通氩气避光搅拌12小时制得APTS-FITC复合物,并在3℃条件下冷藏保存,最后将两部分实验产物与TEOS一起混合搅拌10小时,最后得到CNT/Fe3O4@SiO2(FITC)一维纳米复合材料。该方法的实验条件苛刻与较长反应时间,不具有选择性,荧光素容易被光漂白,会失去探针的作用。2015年,周志钦等人公开了发明专利(CN104979091A)“一种碳包覆磁性纳米粒子”的制备方法和应用。该发明用柑橘果胶、六水氯化铁和尿素溶于水中,充分搅拌使固体完全溶解得到混合溶液,将所得到混合溶液置于180-200℃范围内充分反应,将反应后混合溶液冷却,沉淀,再经分离、洗涤、干燥得碳包裹磁性纳米粒子。该发明对目标分析物无选择性,在后期高温处理的带来的高能耗,也限制了其在实践中的应用。2013年,张腾等人发表了发明专利(CN103545077B)“噁嗪环修饰的Fe3O4@SiO2磁性纳米微球及其制备方法和应用”。该发明将FeCl3·6H2O和乙酸钠溶于乙二醇中,待完全混合后将溶液加入到聚四氟乙烯内嵌的不锈钢高温反应釜中,于100~240℃下反应8h,自然冷却,经乙醇洗涤后真空干燥得到Fe3O4磁性纳米微球。虽然该方法最终成功制得了150~400nm的磁性纳米微球,反应温度较高(100~240℃),不具有选择性。
龚子珊等公开发表的学术期刊文章“磁性纳米粒子的制备及其在重金属离子处理中的应用”(分析测试学报,2014,33(2):231-238)中介绍了磁性纳米粒子普遍的制备方法及部分检测对象。谭丽莎等研究团队报道了“功能化纳米Fe3O4磁性材料的制备及其对水中重金属离子的去除”(化学进展,2013,25(12):2147-2158.)一文中综述了国内外功能化纳米Fe3O4磁性材料的制备方法及其对水中重金属离子去除的研究进展,重点阐述了针对不同重金属离子去除的纳米Fe3O4磁性材料的修饰方法及其应用,并比较了目前应用于水中重金属离子去除的功能化纳米Fe3O4磁性材料制备方法的优缺点。靳艳艳等人发表了“单分散羧基化Fe3O4磁性纳米粒子的制备及表征”(科学通报,2014,59(18):1700-1706.)一文中以油酸铁复合物、油酸、十八烯烃制得的Fe3O4磁性纳米粒子作为基体并成功在表面修饰羧基。
综上所述,制备一种检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体,实现对农药残留的检测。但是,目前尚未见制备Fe3O4@PEG@SiO2人工抗体来检测噻吩磺隆的相关文献和专利的报道。本发明是基于分子印记技术和纳米合成技术,制备的人工抗体中洗脱位于SiO2壳层中的印记分子,SiO2壳层的内部形成具有与印记分子结构、大小和功能基互补的空穴结构,洗脱印记分子的人工抗体具有对目标分析物分子的特异性识别位点,实现对目标分析物分子选择性识别和检测。
发明内容
本发明目的在于针对目前现有技术存在的不足之处,首次在Fe3O4表面修饰聚乙二醇2000层,其表面的聚乙二醇2000分子层中的羟基与目标分子噻吩磺隆中氨基通过氢键相互作用,用TEOS为交联剂,氨水为催化剂在其表面印记噻吩磺隆分子,最终制得到检测噻吩磺隆的Fe3O4@SiO2人工抗体,并将其用于对噻吩磺隆分子的识别与检测。所述的方法为化学合成方法,首先,用FeCl3·6H2O、FeCl2·4H2O和聚乙二醇2000水溶液,调节溶液pH值至碱性,得到表面修饰聚乙二醇2000的Fe3O4纳米粒子;其次,将噻吩磺隆加入到修饰了聚乙二醇2000的Fe3O4纳米粒子溶液中,粒子表面羟基上的氢原子与噻吩磺隆目标分子上带有孤对电子的氮原子两者之间形成氢键结合在磁性纳米粒子周围,再将交联剂正硅酸四乙酯、催化剂氨水、致孔剂十六烷基三甲基溴化铵加到上述混合溶液中去,分阶段升温反应,印记交联聚合,离心、洗涤、分离后得到Fe3O4@PEG@SiO2纳米粒子表面印记目标分子;最后,将所得到印记噻吩磺隆的Fe3O4@PEG@SiO2粒子离心洗涤、超声分散,再加入体积比为1:4的乙酸和丙酮混合液,放置摇床中在室温下震荡反应,将印记分子从印记位点中洗脱至溶液中,离心、超声、再离心、反复洗涤3次,得到具有选择性识别印记分子的芯-壳-壳型Fe3O4@PEG@SiO2人工抗体。
本发明的技术方案是:一种检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备方法,包括在磁性纳米粒子Fe3O4的表面修饰上聚乙二醇2000(Polyethylene glycol 2000,PEG2000),表面包覆纳米SiO2壳层,形成芯-壳-壳结构,其特征在于:所述的人工抗体中洗脱位于SiO2壳层中的印记分子,SiO2壳层的内部形成具有与印记分子结构、大小和功能基互补的空穴结构,洗脱印记分子的人工抗体具有对目标分析物分子的特异性识别位点,实现对目标分析物分子选择性识别和检测,所述的人工抗体制备过程包括如下三个步骤:
1.1 第一步是表面修饰聚乙二醇2000的水溶性磁性纳米粒子Fe3O4的制备:用精度为万分之一的电子天平分别准确称量2.6030 ~ 2.8030 g FeCl3·6H2O和1.8880 ~ 2.9880 gFeCl2·4H2O置于250 mL三口烧瓶中,在氮气气氛下,再将40~60 mL浓度为140~160 g·L-1的聚乙二醇水2000溶液加入到上述烧瓶中,水浴加热至45~55℃,在900 ~ 1100 rpm转速下,加入氨水,将溶液pH值调节在10~12之间反应30~40min,用磁铁将Fe3O4纳米粒子与溶剂进行分离,用去离子水离心洗涤Fe3O4纳米粒子、洗涤的Fe3O4纳米粒子置于250 mL烧瓶中,再将40~60 mL浓度为90~100 g·L-1的聚乙二醇2000水溶液加入到洗涤后的Fe3O4烧瓶中,随后加入去离子水将混合溶液的pH值调节在8 ~ 9之间,最后,超声分散10~20 min,得到表面修饰聚乙二醇2000的Fe3O4纳米粒子;
1.2 第二步是修饰聚乙二醇2000的Fe3O4纳米粒子表面印记目标分子:取上述制得的6mL的表面修饰聚乙二醇2000的Fe3O4纳米粒子溶液,置于100 mL磨口锥形瓶中,再加入20~30mL去离子水稀释,超声分散5 ~10min后,将0.0210~0.0420 mg 噻吩磺隆目标分子置于上述溶液中,超声分散5 ~10min后,静置15~20 min使修饰聚乙二醇2000的Fe3O4纳米粒子表面的羟基可进一步与噻吩磺隆目标分子相互作用,羟基上的氢原子与噻吩磺隆目标分子上带有孤对电子的氮原子两者之间形成氢键结合在磁性纳米粒子周围,将0.5~ 0.7 mL 的交联剂正硅酸四乙酯、适量的催化剂、0.0710~ 0.0920 mg 的十六烷基三甲基溴化铵加到上述混合溶液中去,超声分散5 ~ 10min后,将锥形瓶置于摇床中以转速280~320 rpm、分阶段升温反应,印记交联聚合,首先,在室温下水解聚合反应1.5~2.5h,然后,30~40 ℃下水解聚合反应1.5~2.5h,再次,在40~50 ℃下水解聚合反应1.5~2.5h,最后,在50~70 ℃下水解聚合反应2~3 h,离心洗涤分离后得到Fe3O4@PEG@SiO2纳米粒子表面印记目标分子;
1.3 第三步是检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备:将所得到印记噻吩磺隆的Fe3O4@PEG@SiO2粒子分别均分至三支50 mL离心管中,再用无水工业乙醇超声分散,如此反复3次,最后分别在上述离心洗涤后的Fe3O4@PEG@SiO2粒子中加入5~15 mL去离子水,超声分散,分别再加入10~30mL体积比为1:4的乙酸和丙酮混合液,放置摇床中在室温下以200~300 rpm震荡1h ~ 3h,将印记分子从印记位点中洗脱至溶液中,再将洗脱后的溶液以8000rpm ~ 10000 rpm转速离心5min ~ 10min,离心后的溶液倒去上清液,然后用无水乙醇超声、离心、洗涤3次,最后再用去离子水超声、离心、洗涤3次,得到具有选择性识别印记分子的芯-壳-壳型Fe3O4@PEG@SiO2人工抗体。
作为对现有技术的进一步扩展,所述制备检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体中的印记分子是噻吩磺隆。所述制备检测噻吩磺隆的Fe3O4@SiO2人工抗体中的催化剂是氨水。所述制备检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体中的印记壳层SiO2厚度可控,可以通过调节正硅酸乙酯的量来加以控制。所述制备检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体中的十六烷基三甲基溴化铵是致孔剂。所述制备检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体中的识别位点可以选择性识别相对应的印记分子。
相对于现有技术的有益效果
近年来,Fe3O4@SiO2复合物颗粒合成和应用在材料、生物、医学、化学科学领域吸引了大批研究者的兴趣。Chen, L. 等人发表了“Synthesis of β-Cyclodextrin-ModifiedCellulose Nanocrystals (CNCs)@Fe3O4@SiO2 Superparamagnetic Nanorods”(Acs. Sustain. Chem. Eng., 2014, 2(4): 951-958.)一文中报道的合成了β-环糊精修饰CNC@Fe3O4@SiO2磁性纳米棒拥有良好的吸附性能,能够除去:盐酸普鲁卡因、盐酸丙咪嗪两种化合物。Deng, Y.等人发表了 “Superparamagnetic High-Magnetization Microsphereswith an Fe3O4@SiO2 Core and Perpendicularly Aligned Mesoporous SiO2 Shell forRemoval of Microcystins”(J. Am. Chem. Soc., 2008, 130(1): 28-29.)一文中报道了通过模板剂的溶胶凝胶法合成了一种Fe3O4@SiO2核和一个垂直排列的介孔SiO2壳磁性微球。微球具有高的磁化强度(53.3 emu/g)、高比表面积(365平方米/克),大的孔体积(0.29立方厘米/克),和均匀的孔(2.3 nm)。实现高效率的快速去除微囊藻毒素(> 95%)。Ding,H. L.等人发表了“Fe3O4@SiO2 Core/Shell Nanoparticles: The Silica CoatingRegulations with a Single Core for Different Core Sizes and ShellThicknesses”(Chem. Mater.,2012, 24(23): 4572-4580.)一文中报道了Fe3O4纳米粒子(纳米)的反向微乳液法的涂层方法,得到的Fe3O4的核心/壳纳米粒子。该方法可以适用于不同尺寸的Fe3O4纳米粒子,并避免无芯二氧化硅颗粒的形成。合成均匀的Fe3O4核/壳纳米粒子壳厚度可控制,也可以适用于其他核壳纳米粒子制备。Guo, X.等人发表了“Sulfhydryl-Modified Fe3O4@SiO2 Core/Shell Nanocomposite: Synthesis and ToxicityAssessment in Vitro”(ACS Appl. Mater. Inter., 2015, 7(27): 14983-14991.) 一文中报道了制备巯基修饰的Fe3O4核壳型磁性纳米复合材料,评价其体外毒性,探讨其在生物医学领域的潜在应用。通过简单的溶剂热法合成纳米Fe3O4包覆SiO2通过Stöber法的进一步修饰合成了内消旋-2,3 -二巯基琥珀酸(DMSA)修饰的Fe3O4@SiO2@DMSA纳米粒子。巯基改性的毒性Fe3O4@SiO2核壳纳米颗粒在小鼠成纤维细胞(L-929)中,与物质缺乏的溶血活性,生物相容性好,这表明Fe3O4@SiO2@DMSA纳米复合材料,其适合在生化领域的进一步应用。Hu,H.等人发表了“Ag-Coated Fe3O4@SiO2 Three-Ply Composite Microspheres: Synthesis,Characterization, and Application in Detecting Melamine with Their Surface-Enhanced Raman Scattering”(J. Phys. Chem. C, 2010, 114(17): 7738-7742.)一文中报道了采用银镜反应,将平均粒径为20 nm的Ag纳米颗粒分散在Fe3O4@SiO2复合微球的表面,利用表面增强拉曼技术来检测三聚氰胺浓度。Li, X. 等人发表了“Fe3O4@SiO2@TiO2@PtHierarchical Core–Shell Microspheres: Controlled Synthesis, EnhancedDegradation System, and Rapid Magnetic Separation to Recycle”(Cryst. Growth Des.,2014, 14(11): 5506-5511.) 一文中报道了成功地合成了由SiO2包覆的Fe3O4的核磁性复合微球,SiO2壳层由一个有序的TiO2层次结构的外壳,和Pt纳米颗粒层分散在TiO2纳米片表面,具有高效降解罗丹明B的功能。Mohapatra, S.等人发表了“Design of Fe3O4@SiO2@Carbon Quantum Dot Based Nanostructure for Fluorescence Sensing, MagneticSeparation, and Live Cell Imaging of Fluoride Ion”(Langmuir, 2015, 31(29):8111-8120.) 一文中报道了一种可重复使用的化学识别和荧光信号检测受体的Fe3O4@SiO2@CQD氟传感器的方法。Morel, A.-L.等人发表了“Sonochemical Approach to theSynthesis of Fe3O4@SiO2 Core−Shell Nanoparticles with Tunable Properties”(ACS Nano, 2008, (5): 847-856.) 一文中报道了一种快速的超声化学法合成单分散的、壳厚度可控的Fe3O4@SiO2磁纳米粒子的方法。Palani, A. 等人发表了“Selective Enrichmentof Cysteine-Containing Peptides Using SPDP-Functionalized SuperparamagneticFe3O4@SiO2 Nanoparticles: Application to Comprehensive Proteomic Profiling”(J. Proteome Res., 2008, 7(8): 3591-3596.)一文中报道了一种超顺磁性Fe3O4@SiO2核壳纳米粒子(约30 nm直径),其表面修饰了硫醇特定官能团,表现出对半胱氨酰肽较高的捕获效率。Patil, U. S. 人发表了“Labeling Primary Amine Groups in Peptides andProteins with N-Hydroxysuccinimidyl Ester Modified Fe3O4@SiO2 NanoparticlesContaining Cleavable Disulfide-Bond Linkers”(Bioconjugate Chem., 2013, 24(9):1562-1569.)一文中报道了超顺磁性二氧化硅包覆氧化铁表面(Fe3O4@SiO2)纳米粒子与二硫键连接的N-羟基琥珀酰亚胺酯基官能化(NHS)为了建立一个标记的肽/蛋白质的伯胺的方法,使NHS酯包覆Fe3O4纳米粒子的成为一个潜在的标记探针,用于研究活细胞表面上的蛋白质。Qiu, H.等人发表了“Novel Fe3O4@ZnO@@mSiO2 Nanocarrier for Targeted DrugDelivery and Controllable Release with Microwave Irradiation”(J. Phys. Chem. C, 2014, 118(27):14929-14937.)一文中报道制备了具有磁性Fe3O4芯的介孔二氧化硅壳和具有核壳结构的ZnO中间层的新型复合纳米复合材料。这种新型的核壳结构的纳米载体——作为药物载体研究的存储与化疗药物依托泊苷的控制释放性能。介孔纳米载体具有较高的比表面积(643.9平方米/克),较大的孔隙体积(0.32立方厘米/克)便于药物分子的吸附,并具有高的饱和磁化强度值(56.8 emu/g),外磁场下便于靶向药物,ZnO层作为一种具有优良的微波热响应特性的微波吸收体,触发药物释放。Shao, M.等人发表了“Preparation of Fe3O4@SiO2@Layered Double Hydroxide Core–Shell Microspheresfor Magnetic Separation of Proteins”(J. Am. Chem. Soc.,2012, 134(2): 1071-1077.)一文中制备了一种Fe3O4@SiO2@NiAl-LDH微球三维核心–壳结构,拥有花状形态,比表面积大(83平方米/克),和均匀的孔道(4.3nm)。可用于重组蛋白纯化的实践,以及在各种包括药和生物传感器等生物医学领域应用的潜力。Shen, J. 等人发表了“MultifunctionalFe3O4 @Ag/SiO2/Au Core–Shell Microspheres as a Novel SERS-Activity Label viaLong-Range Plasmon Coupling" (Langmuir, 2013, 29(2): 690-695.)一文中报道了新颖的多功能的四氧化三铁的金/金/壳核壳微球,显示远程Ag/Au的等离子体激元转移,导致增强拉曼散射。Tong, L.等人发表了“Luminescent and Magnetic Properties of Fe3O4@SiO2 @Y2O3:Eu3+ Composites with Core–Shell Structure”(J. Phys. Chem. C, 2012,116(12): 7153-7157.)一文中报道了采用简单溶剂热法制备了多功能Fe3O4@SiO2@Y2O3:Eu3 +复合材料。Wang, H.等人发表了“Rapid Decolorization of Phenolic Azo Dyes byImmobilized Laccase with Fe3O4@SiO2 Nanoparticles as Support”( Ind. Eng. Chem. Res., 2013, 52(12): 4401-4407.)一文中报道了通过戊二醛偶联,用粒径在30 nm以下的Fe3O4@SiO2纳米颗粒作为漆酶固定化载体,讨论了固定化漆酶对酚类偶氮染料脱色的可能机理。Wehner, T.等人发表了“Superparamagnetic Luminescent MOF@Fe3O4@SiO2Composite Particles for Signal Augmentation by Magnetic Harvesting asPotential Water Detectors”(ACS. Appl. Mater. Inter, 2016, 8(8): 5445-5452.)一文中报道了一种涂有发光的金属有机骨架(MOFs)的超顺磁性核壳型的Fe3O4/SiO2复合微粒的复杂粒子系统合成方法。Zhang, W. 等人发表了“Tetraazacalix[2]arence[2]triazineCoated Fe3O4@SiO2 Magnetic Nanoparticles for Simultaneous Dispersive SolidPhase Extraction and Determination of Trace Multitarget Analytes” (Anal. Chem., 2016, 88(21): 10523-10532.)一文中报道了一种有机化合物涂层的Fe3O4@SiO2磁性纳米粒子的制备,该粒子可以同时进行多目标目标分析物的固相萃取分离和测定。Zhao,P. 等人发表了“Effect of the Structure and Length of Flexible Chains onDendrimers Grafted Fe3O4@SiO2/PAMAM Magnetic Nanocarriers for LipaseImmobilization” (Acs. Sustain. Chem. Eng., 2016, 4(12): 6382-6390.)一文中报道了用三种不同的胺试剂接枝树枝状大分子获得各种Fe3O4@SiO2/磁性纳米载体与不同代的PAMAM,广泛应用于生物大分子酶和蛋白质的固定化、催化载体、基因治疗和药物输送。Zhu,Y. 等人发表了“Folate-Conjugated Fe3O4@SiO2 Hollow Mesoporous Spheres forTargeted Anticancer Drug Delivery” (J. Phys. Chem. C, 2010, 114(39): 16382-16388.)一文中报道了开发了一个有针对性的抗癌药物传递系统的基础上叶酸共轭拨浪鼓型Fe3O4中空中空球结合受体介导的靶向和磁靶向。Zhu, Y. 等人发表了“An EfficientRoute to Rattle-Type Fe3O4@SiO2 Hollow Mesoporous Spheres Using ColloidalCarbon Spheres Templates” (Chem. Mater., 2009, 21(12): 2547-2553.)一文中报道了以胶体碳球为模板成功制备了具有大腔体的拨浪鼓型空心SiO2介孔微球。球体具有良好的单分散性和几乎一致的尺寸(约900 nm)。介孔二氧化硅壳的厚度约100纳米,直径约为100 nm的Fe3O4颗粒仅包裹在每个中空的介孔二氧化硅微球中,Fe3O4@SiO2中空介孔球具有较高的载药量和缓释性能。2015年,杨鑫等人公开了发明专利(CN105254827A)“一种分离烟碱农药哌虫啶的磁性纳米分子印记聚合物”的制备方法,该发明利用三氯化铁、乙二醇、无水乙酸钠放置在高温反应釜中,在温度为180 ~ 220°C条件下反应l h ~ 13 h,停止反应静置15~25min,制得Fe3O4磁性纳米粒子,然后,用盐酸和柠檬酸三钠对其表面进行改性,加入正硅酸乙酯和氨水水解后合成了Fe3O4@SiO2,然后再用硅烷化试剂3-(三甲氧基甲硅烷基)丙基丙烯酸酯水解、用无水甲苯、乙醇洗涤得到Fe3O4@SiO2@C=C复合物颗粒,最后加入目标分子哌虫啶、交联剂乙二醇二甲基丙烯酸酯和引发剂偶氮二异丁晴聚合,得到印记哌虫啶的Fe3O4@SiO2@C=C分子印记聚合物,该发明对哌虫啶的最大饱和吸附量为17.305±0.403mg/g,是非分子印记最大饱和吸附量的2.0倍。该方法过程繁琐,饱和结合量小,吸附动力学慢,高分子聚合物刚性弱,易坍塌,有效识别位点数量减少,制备过程用甲苯溶剂洗涤,对环境不友好。2016年,王永强等人公开了发明专利(CN105832669A)“一种Fe3O4@SiO2蛋黄-蛋壳结构中空复合微球的制备方法及应用”。该发明利用FeCl3·6H2O、尿素、柠檬酸三钠的水溶液在加入聚丙烯酸钠的条件下加热制得Fe3O4纳米微球。该方法在后续步骤中以正硅酸乙酯(Tetraethyl orthosilicate,TEOS)作为硅源,经浓盐酸腐蚀后还原得到Fe3O4@SiO2芯-壳结构微球,用于药物的缓释释放。由于浓盐酸的腐蚀性与环境危害性,该方法在实际推广过程中将受到一定限制。2016年,王娜等人公开了发明专利(CN106118630A)“磁光双功能CNT/Fe3O4@SiO2(FITC)一维纳米复合材料的制备方法”。该方法将多壁碳纳米管、乙酰丙酮铁、三甘醇加热1小时后制得CNT/Fe3O4,随后利用异硫氰酸荧光素(Fluoresceinisothiocyanate,FITC)与3-氨丙基三乙氧基硅烷在乙醇中通氩气避光搅拌12小时制得APTS-FITC复合物,并在3℃条件下冷藏保存,最后将两部分实验产物与TEOS一起混合搅拌10小时,最后得到CNT/Fe3O4@SiO2(FITC)一维纳米复合材料。该方法的实验条件苛刻与较长反应时间,不具有选择性,荧光素容易被光漂白,会失去探针的作用。
尽管这些文献报道的有些是针对不同目标分析物识别和检测的制备方法,所述的方法中没有携带专识性的基团,选择性差,即使是表面修饰了功能基团仅仅是针对其他目标分析物,且不具有选择性,未见检测噻吩磺隆目标分析物的报道,更未涉及到利用表面修饰聚乙二醇2000的Fe3O4@PEG@SiO2人工抗体对噻吩磺隆检测报道。因此,制备具有高选择性和高灵敏地检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的方法,实现对噻吩磺隆分子识别和检测有其必要性。
附图说明
图1一种检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备方法示意图。
图2 Fe3O4磁性纳米粒子XRD图。
图3表面修饰了聚乙二醇2000的Fe3O4磁性粒子的傅里叶红外光谱图。
图4 Fe3O4@PEG@SiO2人工抗体SEM图。
图5 Fe3O4磁性粒子表面的聚乙二醇2000在混合溶液中与目标分析物通过氢键相互作用。
图6 聚乙二醇2000水溶液中加入不同浓度的噻吩磺隆时紫外-可见光谱图。
图7 印记的Fe3O4@PEG@SiO2人工抗体对噻吩磺隆(□)吸附等温线,同样条件下合成的非印记的Fe3O4@PEG@SiO2人工抗体对噻吩磺隆(○)吸附等温线。
图8 印记的Fe3O4@PEG@SiO2人工抗体对噻吩磺隆(□)动力学曲线,同样条件下合成的非印记Fe3O4@PEG@SiO2人工抗体对噻吩磺隆(○)动力学曲线。
根据附图进一步解释具体实施方式
图1中首先在碱性条件下用FeCl3·6H2O、FeCl2·4H2O和聚乙二醇2000制备Fe3O4磁性粒子,处理洗净后的Fe3O4磁性粒子中加入一定浓度的聚乙二醇2000水溶液,调节溶液pH值,超声分散后得到表面修饰了聚乙二醇2000的Fe3O4磁性粒子,然后加入目标分子噻吩磺隆,交联剂正硅酸四乙酯、催化剂氨水、致孔剂十六烷基三甲基溴化铵加到上述混合溶液中去,超声分散、从室温开始分阶段升温水解、反应聚合、离心洗涤、分离后得到Fe3O4纳米粒子表面印记目标分子,再加入乙酸和丙酮混合液,将噻吩磺隆印记分子从印记位点中洗脱至溶液中,得到具有选择性识别噻吩磺隆印记分子的芯-壳-壳型Fe3O4@PEG@SiO2人工抗体。
图2 Fe3O4磁性纳米粒子XRD图,经JDPS#75-0033标准卡对照,2θ分别为18.31度对应的晶面指数为(1 1 1),30.12度对应的晶面指数为(2 2 0),35.48度对应的晶面指数为(3 1 1),43.12度对应的晶面指数为(4 0 0),57.03度对应的晶面指数为(5 1 1),62.63度对应的晶面指数为(4 4 0),上述晶面显示的是Fe3O4特征晶面指数。
图3中表面修饰了聚乙二醇2000的Fe3O4磁性粒子红外光谱图,-O-H伸缩振动波数在3450cm-1处,C-H弯曲振动波数在1417cm-1处,纳米颗粒在586 cm-1附近有较大的Fe-O键特征吸收峰,且经修饰后该特征吸收峰未发生明显变化。
图4图中可以看出制备印记了噻吩磺隆Fe3O4@PEG@SiO2人工抗体在SEM下形貌为颗粒状,尺度为纳米级。
图5是分散在水溶液中的聚乙二醇2000表面的羟基与噻吩磺隆中的酰胺基团中氨基上氮原子的孤对电子相互作用,形成非共价键——氢键,从而可以导致模板分子噻吩磺隆进入识别位点与功能单体聚乙二醇2000发生作用。
图6是一定浓度的聚乙二醇2000水溶液中,从下至上依次是加入浓度为0M,1×10- 5M,2×10-5 M,3×10-5 M,4×10-5 M,5×10-5 M,6×10-5 M,由于聚乙二醇与噻吩磺隆通过分子间氢键相互作用,紫外光谱波长明显朝着长波长方向移动,发生了红移。
图7 20mL浓度为1×10-5M,2×10-5M,3×10-5M,4×10-5M,5×10-5M,6×10-5M的噻吩磺隆加到20mg的印记的Fe3O4@PEG@SiO2人工抗体中的(□)吸附等温线。吸附平衡时对应的噻吩磺隆最大浓度为5×10-5M,最大饱和结合量为41.28mg/g。而非印记的Fe3O4@PEG@SiO2人工抗体对噻吩磺隆(○)最大饱和结合量为7.72mg/g,印记的人工抗体最大饱和结合量是非印记的人工抗体最大饱和结合量5.34倍。
图8是20mg的印记的Fe3O4@PEG@SiO2人工抗体中的(□)对浓度为5×10-5M噻吩磺隆吸附动力学曲线,前30分种内,吸附速率为0.45mg/g·min,同样条件下而非印记的Fe3O4@PEG@SiO2人工抗体对噻吩磺隆(○)吸附动力学曲线,前30分种内,吸附速率为0.13mg/g·min,前者是后者的3.46倍。
具体实施方式
一种检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备方法,包括在Fe3O4磁性纳米粒子的表面修饰聚乙二醇2000(Polyethylene glycol 2000,PEG 2000),其表面包覆纳米SiO2壳层,形成芯-壳-壳结构,其特征在于:所述的人工抗体中洗脱位于SiO2壳层中的印记分子,SiO2壳层的内部形成具有与印记分子结构、大小和功能基互补的空穴结构,洗脱印记分子的人工抗体具有对目标分析物分子的特异性识别位点,实现对目标分析物分子选择性识别和检测,所述的人工抗体制备过程包括如下三个步骤:
1.1 第一步是表面修饰聚乙二醇2000的水溶性磁性纳米粒子Fe3O4的制备:用精度为万分之一的电子天平分别准确称量2.6030 ~ 2.8030 g FeCl3·6H2O和1.8880 ~ 2.9880 gFeCl2·4H2O置于250 mL三口烧瓶中,在氮气气氛下,再将40~60 mL浓度为140~160 g·L-1的聚乙二醇水2000溶液加入到上述烧瓶中,水浴加热至45~55℃,在900 ~ 1100 rpm转速下,加入氨水,将溶液pH值调节在10~12之间反应30~40min,用磁铁将Fe3O4纳米粒子与溶剂进行分离,用去离子水离心洗涤Fe3O4纳米粒子、洗涤的Fe3O4纳米粒子置于250 mL烧瓶中,再将40~60 mL浓度为90~100 g·L-1的聚乙二醇2000水溶液加入到洗涤后的Fe3O4烧瓶中,随后加入去离子水将混合溶液的pH值调节在8 ~ 9之间,最后,超声分散10~20 min,得到表面修饰聚乙二醇2000的Fe3O4纳米粒子;
1.2 第二步是修饰聚乙二醇2000的Fe3O4纳米粒子表面印记目标分子:取上述制得的6mL的表面修饰聚乙二醇2000的Fe3O4纳米粒子溶液,置于100 mL磨口锥形瓶中,再加入20~30mL去离子水稀释,超声分散5 ~10min后,将0.0210~0.0420 mg 噻吩磺隆目标分子置于上述溶液中,超声分散5 ~10min后,静置15~20 min使修饰聚乙二醇2000的Fe3O4纳米粒子表面的羟基可进一步与噻吩磺隆目标分子相互作用,羟基上的氢原子与噻吩磺隆目标分子上带有孤对电子的氮原子两者之间形成氢键结合在磁性纳米粒子周围,将0.5~ 0.7 mL 的交联剂正硅酸四乙酯、适量的催化剂、0.0710~ 0.0920 mg 的十六烷基三甲基溴化铵加到上述混合溶液中去,超声分散5 ~ 10min后,将锥形瓶置于摇床中以转速280~320 rpm、分阶段升温反应,印记交联聚合,首先,在室温下水解聚合反应1.5~2.5h,然后,30~40 ℃下水解聚合反应1.5~2.5h,再次,在40~50 ℃下水解聚合反应1.5~2.5h,最后,在50~70 ℃下水解聚合反应2~3 h,离心洗涤分离后得到Fe3O4@PEG@SiO2纳米粒子表面印记目标分子;
1.3 第三步是检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备:将所得到印记噻吩磺隆的Fe3O4@PEG@SiO2粒子分别均分至三支50 mL离心管中,再用无水工业乙醇超声分散,如此反复3次,最后分别在上述离心洗涤后的Fe3O4@PEG@SiO2粒子中加入5~15 mL去离子水,超声分散,分别再加入10~30mL体积比为1:4的乙酸和丙酮混合液,放置摇床中在室温下以200~300 rpm震荡1h ~ 3h,将印记分子从印记位点中洗脱至溶液中,再将洗脱后的溶液以8000rpm ~ 10000 rpm转速离心5min ~ 10min,离心后的溶液倒去上清液,然后用无水乙醇超声、离心、洗涤3次,最后再用去离子水超声、离心、洗涤3次,得到具有选择性识别印记分子的芯-壳-壳型Fe3O4@PEG@SiO2人工抗体。
实施例:利用分子印记技术和纳米合成技术,制备检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体,采用三步反应得到上述人工抗体:
1.1 第一步是表面修饰聚乙二醇2000的水溶性磁性纳米粒子Fe3O4的制备:用精度为万分之一的电子天平分别准确称量2.7030 g FeCl3·6H2O和1.9880 g FeCl2·4H2O置于250 mL三口烧瓶中,在氮气气氛下,再将50 mL浓度为150 g·L-1的聚乙二醇水2000溶液加入到上述烧瓶中,水浴加热至50℃,在1000 rpm转速下,加入氨水,将溶液pH值调节至11,反应35 min,用磁铁将Fe3O4纳米粒子与溶剂进行分离,用去离子水离心洗涤Fe3O4纳米粒子、洗涤的Fe3O4纳米粒子置于250 mL烧瓶中,再将50 mL浓度为95 g·L-1的聚乙二醇2000水溶液加入到洗涤后的Fe3O4烧瓶中,随后加入去离子水将混合溶液的pH值调节至9,最后,超声分散15 min,得到表面修饰聚乙二醇2000的Fe3O4纳米粒子;
1.2 第二步是修饰聚乙二醇2000的Fe3O4纳米粒子表面印记目标分子:取上述制得的6mL的表面修饰聚乙二醇2000的Fe3O4纳米粒子溶液,置于100 mL磨口锥形瓶中,再加入25 mL去离子水稀释,超声分散8 min后,将0.0310 mg 噻吩磺隆目标分子置于上述溶液中,超声分散8 min后,静置16 min使修饰聚乙二醇2000的Fe3O4纳米粒子表面的羟基可进一步与噻吩磺隆目标分子相互作用,羟基上的氢原子与噻吩磺隆目标分子上带有孤对电子的氮原子两者之间形成氢键结合在磁性纳米粒子周围,将0.6 mL 的交联剂正硅酸四乙酯、适量的催化剂、0.0810 mg 的十六烷基三甲基溴化铵加到上述混合溶液中去,超声分散6 min后,将锥形瓶置于摇床中以转速300 rpm、分阶段升温反应,印记交联聚合,首先,在室温下水解聚合反应2 h,然后,35 ℃下水解聚合反应2 h,再次,在45 ℃下水解聚合反应2 h,最后,在60℃下水解聚合反应2.5 h,离心洗涤分离后得到Fe3O4@PEG@SiO2纳米粒子表面印记目标分子;
1.3 第三步是检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备:将所得到印记噻吩磺隆的Fe3O4@PEG@SiO2粒子分别均分至三支50 mL离心管中,再用无水工业乙醇超声分散,如此反复3次,最后分别在上述离心洗涤后的Fe3O4@PEG@SiO2粒子中加入10 mL去离子水,超声分散,分别再加入20 mL体积比为1:4的乙酸和丙酮混合液,放置摇床中在室温下以250 rpm震荡2 h,将印记分子从印记位点中洗脱至溶液中,再将洗脱后的溶液以9000 rpm转速离心8 min,离心后的溶液倒去上清液,然后用无水乙醇超声、离心、洗涤3次,最后再用去离子水超声、离心、洗涤3次,得到具有选择性识别印记分子的芯-壳-壳型Fe3O4@PEG@SiO2人工抗体。
Claims (6)
1.一种检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备方法,包括在Fe3O4磁性纳米粒子的表面修饰聚乙二醇2000(Polyethylene glycol 2000,PEG 2000),其表面包覆纳米SiO2壳层,形成芯-壳-壳结构,其特征在于:所述的人工抗体中洗脱位于SiO2壳层中的印记分子,SiO2壳层的内部形成具有与印记分子结构、大小和功能基互补的空穴结构,洗脱印记分子的人工抗体具有对目标分析物分子的特异性识别位点,实现对目标分析物分子选择性识别和检测,所述的人工抗体制备过程包括如下三个步骤:
1.1 第一步是表面修饰聚乙二醇2000的水溶性磁性纳米粒子Fe3O4的制备:用精度为万分之一的电子天平分别准确称量2.6030 ~ 2.8030 g FeCl3·6H2O和1.8880 ~ 2.9880 gFeCl2·4H2O置于250 mL三口烧瓶中,在氮气气氛下,再将40~60 mL浓度为140~160 g·L-1的聚乙二醇水2000溶液加入到上述烧瓶中,水浴加热至45~55℃,在900 ~ 1100 rpm转速下,加入氨水,将溶液pH值调节在10~12之间反应30~40min,用磁铁将Fe3O4纳米粒子与溶剂进行分离,用去离子水离心洗涤Fe3O4纳米粒子、洗涤的Fe3O4纳米粒子置于250 mL烧瓶中,再将40~60 mL浓度为90~100 g·L-1的聚乙二醇2000水溶液加入到洗涤后的Fe3O4烧瓶中,随后加入去离子水将混合溶液的pH值调节在8 ~ 9之间,最后,超声分散10~20 min,得到表面修饰聚乙二醇2000的Fe3O4纳米粒子;
1.2 第二步是修饰聚乙二醇2000的Fe3O4纳米粒子表面印记目标分子:取上述制得的6mL的表面修饰聚乙二醇2000的Fe3O4纳米粒子溶液,置于100 mL磨口锥形瓶中,再加入20~30mL去离子水稀释,超声分散5 ~10min后,将0.0210~0.0420 mg 噻吩磺隆目标分子置于上述溶液中,超声分散5 ~10min后,静置15~20 min使修饰聚乙二醇2000的Fe3O4纳米粒子表面的羟基可进一步与噻吩磺隆目标分子相互作用,羟基上的氢原子与噻吩磺隆目标分子上带有孤对电子的氮原子两者之间形成氢键结合在磁性纳米粒子周围,将0.5~ 0.7 mL 的交联剂正硅酸四乙酯、适量的催化剂、0.0710~ 0.0920 mg 的十六烷基三甲基溴化铵加到上述混合溶液中去,超声分散5 ~ 10min后,将锥形瓶置于摇床中以转速280~320 rpm、分阶段升温反应,印记交联聚合,首先,在室温下水解聚合反应1.5~2.5h,然后,30~40 ℃下水解聚合反应1.5~2.5h,再次,在40~50 ℃下水解聚合反应1.5~2.5h,最后,在50~70 ℃下水解聚合反应2~3 h,离心洗涤分离后得到Fe3O4@PEG@SiO2纳米粒子表面印记目标分子;
1.3 第三步是检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备:将所得到印记噻吩磺隆的Fe3O4@PEG@SiO2粒子分别均分至三支50 mL离心管中,再用无水工业乙醇超声分散,如此反复3次,最后分别在上述离心洗涤后的Fe3O4@PEG@SiO2粒子中加入5~15 mL去离子水,超声分散,分别再加入10~30mL体积比为1:4的乙酸和丙酮混合液,放置摇床中在室温下以200~300 rpm震荡1h ~ 3h,将印记分子从印记位点中洗脱至溶液中,再将洗脱后的溶液以8000rpm ~ 10000 rpm转速离心5min ~ 10min,离心后的溶液倒去上清液,然后用无水乙醇超声、离心、洗涤3次,最后再用去离子水超声、离心、洗涤3次,得到具有选择性识别印记分子的芯-壳-壳型Fe3O4@PEG@SiO2人工抗体。
2.根据权利要求1所述的一种检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备方法,其特征是:所述制备检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体中的印记分子是噻吩磺隆。
3.根据权利要求1所述的一种检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备方法,其特征是:所述制备检测噻吩磺隆的Fe3O4@SiO2人工抗体中的催化剂是氨水。
4.根据权利要求1所述的一种检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备方法,其特征是:所述制备检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体中的印记壳层SiO2厚度可控,可以通过调节正硅酸乙酯的量来加以控制。
5.根据权利要求1所述的一种检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备方法,其特征是:所述制备检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体中的十六烷基三甲基溴化铵是致孔剂。
6.根据权利要求1所述的一种检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体的制备方法,其特征是:所述制备检测噻吩磺隆的Fe3O4@PEG@SiO2人工抗体中的识别位点可以选择性识别相对应的印记分子。
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