CN107216422B - 一种黄曲霉毒素限进介质-分子印迹分离介质的制备方法及应用 - Google Patents
一种黄曲霉毒素限进介质-分子印迹分离介质的制备方法及应用 Download PDFInfo
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Abstract
本发明属于分离介质制备技术领域,特别涉及一种限进介质和印迹聚合物结合的分离介质,并以此作为固相萃取柱的填料应用于复杂基质中黄曲霉毒素选择性的富集、分离。所述印迹聚合物以黄曲霉毒素的结构类似物作为替代模板,将印迹聚合物与限进介质相结合,制得黄曲霉毒素的限进介质‑分子印迹分离介质。本发明所制备的分离介质兼具对黄曲霉毒素选择性的富集和对蛋白质大分子的排阻,将其应用于复杂基质的样品前处理中,达到较好的净化、富集效果,有广阔的应用前景。
Description
技术领域
本发明属于分离介质制备技术领域,特别涉及一种适用于复杂基质中对黄曲霉毒素具有特异性分子识别能力的限进介质-印迹聚合物的制备方法及其应用。
背景技术
黄曲霉毒素(Aflatoxins,AF)主要由黄曲霉和寄生曲霉在霉变过程中产生的次生代谢产物,具有广泛的污染范围特别是对农产品的污染,如大米、小麦、玉米、大豆、高粱和花生等粮食作物,既可在各种农作物、植物及其产品的储藏、制备与加工过程中产生,也会对以我国传统的植物性中药材及其制品造成污染。
黄曲霉毒素对人类和动物的毒性极大而且毒害作用多样。例如,暴露在黄曲霉毒素中会损害中枢神经,心血管和呼吸***,引起急性和慢性毒性或死亡。动物一旦摄入了被真菌毒素污染的饲料,其乳、肉、蛋中能积累、残留这些真菌毒素。通过食物链的转化,这些被污染的食物进入人或动物的的体内, 会导致突变,畸形和降低免疫力等。
由于黄曲霉毒素对各类食物的严重污染和对人类健康的严重威胁,已成为全世界关注的焦点,因此迫切需要建立一种简单、快速、准确、灵敏、特异、经济的检测方法去控制和监测真菌毒素的污染,保障人类的身体健康。目前已建立的分析检测方法主要有薄层色谱法、免疫亲和柱-高效液相色谱法和紫外、荧光、质谱检测器以及酶联免疫吸附试验。但真菌毒素多为痕量检测,且受污染的对象基质多比较复杂,增大了检测难度,因此其前处理技术的好坏在很大程度上决定了检测结果的准确与否。
目前黄曲霉毒素的前处理除了液液萃取、固相萃取等传统方法外,免疫亲合柱是常用的样品前处理方法,但免疫亲合柱的净化效果容易受样品基质、pH、溶剂、盐浓度等的影响,同时其价格昂贵,难于重复使用,不适宜大面积推广使用,很大程度上限制了黄曲霉毒素检测的普及。
新近发展起来的分子印迹聚合物作为固相萃取柱的填料,以其高选择性和特异性的吸附能力、重复性高、检测线低、又不易受样品基体的干扰的特点,正被逐渐应用到食品中对痕量有毒有害物质的前处理过程中,弥补了传统固相萃取和免疫亲和柱的不足。
由于真菌毒素有较强毒性和广泛传播性对试验操作人员有很强的毒性作用,毒素获取困难,价格昂贵。用毒素本身作模板分子会增加实验的成本和操作的危险性,所以研究用毒素的结构类似物作为替代模板合成印迹聚合物,避免了其中的模板因未洗脱完全,对目标分析物的检测造成干扰,降低对实验人员的伤害,毒素的特异性吸附不会造成太大影响且符合环境友好的要求。本发明中采用黄曲霉毒素的结构类似物作为替代模板合成印迹聚合物。
当用固相萃取富集分离复杂基质中的小分子毒素目标物时,其中的生物大分子如蛋白质、油脂分子等,遇到疏水性反相固相萃取填料时会发生变性,变性后的大分子物质会吸附在填料的表面,严重干扰对目标物的分析测定。而限进介质作为固相萃取的吸附剂在一定程度上克服了上述不足。限进介质是一种外层具有亲水性,内层具有富集能力的多功能材料,只要对其外表面进行适当的亲水性修饰,就可保证生物大分子在吸附剂的外表面不会发生不可逆的变性和吸附,实现对大分子的排阻。本发明把限进介质与分子印迹结合起来构建新的分离体系,使其兼具对小分子选择性的富集和对大分子的排阻,将具有极为广阔的应用前景,目前尚未见报道。
发明内容
针对复杂基质中存在的痕量黄曲霉毒素,样品前处理采用传统的液液溶剂萃取效率低,使用商品化的免疫亲和柱净化效果容易受样品基质、pH、溶剂、盐浓度等的影响,同时其价格昂贵,难于重复使用等问题,本发明采用悬浮聚合的方法,拟解决的技术问题是提供一种适用于复杂基质中对黄曲霉毒素具有特异性分子识别能力的限进介质-印迹聚合物的制备方法。
本发明实现上述目的的技术解决方案如下:
在聚乙烯醇溶液中加入替代模板、功能单体、能水解的单体、交联剂、引发剂,加热搅拌反应,结束后用水洗去聚乙烯醇,得到印迹聚合物;酸性条件下水解,得到表面羟基修饰的限进介质-印迹聚合物,洗去模板分子得到对黄曲霉毒素有选择性吸附的分离介质。所述的替代模板为7-乙酰氧基-4-甲基香豆素、6-甲基-4-苯基-2-色满酮、香豆素-3-羧酸乙酯、香豆素-3-甲酸乙酯;所述的功能单体为甲基丙烯酸甲酯、甲基丙烯酸丁酯、丙烯酸、甲基丙烯酸、丙烯酰胺、氯甲基苯乙烯;所述的能水解的单体为甲基丙烯酸缩水甘油酯、丙烯酸缩水甘油酯;所述的交联剂为乙二醇二甲醇丙烯酸酯、二乙烯基苯;所述的引发剂为偶氮二异丁腈、过氧化苯酰;
所述分离介质通过如下步骤制得:
(1)黄曲霉毒素替代分子印迹聚合物的合成:在聚乙烯醇溶液中加入替代模板、功能单体、能水解的单体、交联剂、引发剂,加热搅拌反应,结束后用水洗去聚乙烯醇,得到印迹聚合物;所述的替代模板为7-乙酰氧基-4-甲基香豆素、6-甲基-4-苯基-2-色满酮、香豆素-3-羧酸乙酯、香豆素-3-甲酸乙酯;所述的功能单体为甲基丙烯酸甲酯、甲基丙烯酸丁酯、丙烯酸、甲基丙烯酸、丙烯酰胺、氯甲基苯乙烯;所述的能水解的单体为甲基丙烯酸缩水甘油酯、丙烯酸缩水甘油酯;所述的交联剂为乙二醇二甲醇丙烯酸酯、二乙烯基苯;所述的引发剂为偶氮二异丁腈、过氧化苯酰;
(2)限进介质-分子印迹聚合物的合成:把(1)制得的印迹聚合物加入到酸的水溶液(1:9, V/V)中水解,反应结束后,抽滤,洗涤,晾干,得到限进介质-分子印迹聚合物;
(3)模板分子的洗脱:把(2)制得的聚合物在索氏提取器用有机溶剂-酸的混合物抽提,洗去模板分子,直到不能检测出模板分子为止,再用有机溶剂洗去残留的酸,真空干燥至恒重,得到可以在复杂基质体系中对黄曲霉毒素有选择性吸附的限进介质-印迹聚合物。
同时用同样比例和方法制取非印迹聚合物(不加模板分子)。
步骤(1)中所述的替代模板、功能单体、能水解的单体的比例为1: 4~9: 3~8。
步骤(1)中所述的模板分子和交联剂的比例为1:10~100。
步骤(1)中所述的引发剂的比例为0.5%~5%,反应温度为50℃~100℃,反应时间为2小时~48小时。
步骤(2)中,所述的酸为高氯酸。
步骤(3)中,所述的有机溶剂为甲醇、乙醇、乙腈,所述的酸为乙酸,印迹聚合物用有机溶剂和酸混合液去除模板分子,处理后得到对黄曲霉毒素有选择性吸附的限进介质-印迹聚合物。
附图说明
图1限进介质-分子印迹聚合物(RAM-MIP)的电镜扫描图;
图2限进介质-分子印迹聚合物(RAM-MIP)的粒径分布图;
图3 限进介质-分子印迹聚合物(RAM-MIP)、限进介质-非印迹聚合物(RAM-NIP)和分子印迹聚合物(MIP)的热力学实验;
具体说明
实施例1
将1.0 mmol的6-甲基-4-苯基-2-色满酮加入装有10mL的三氯甲烷的烧杯中,超声使其完全溶解, 然后加入6.0 mmol α-甲基丙烯酸。用滴液漏斗将其滴加到100mL的聚乙烯醇悬浮液中,充分磁力搅拌2 小时后,再分别加入6.0 mmol 甲基丙烯酸缩水甘油酯、40mmol乙二醇二甲基丙烯酸酯和1.0%的偶氮二异丁腈。超声5min,待溶液混合均匀,慢慢滴加到三口瓶中,机械搅拌,置于75℃恒温油浴中,反应 8h,高速离心得反应产物。
将聚合物放入沸水中重复洗涤6-10次,直至完全除去聚合物表面的聚乙烯醇。然后用甲醇/乙酸(9∶1,体积/体积)与甲醇清洗反应产物,直到无模板分子洗出为止。晾干后于40℃下真空干燥24h,得到能够进一步水解的分子印迹聚合物。
将分子印迹聚合物放入到50 mL高氯酸水溶液(1:9, V/V)中磁力搅拌24 h,然后抽滤,将聚合物依次在200 mL的乙醇、丙酮、***中不断回流洗脱,最后抽虑,晾干,得到限进介质-分子印迹聚合物。
限进介质-非印迹聚合物的制备方法,除不加模板分子外,其他同上。
实施例 2
将1.0 mmol的 6-甲基-4-苯基-2-色满酮加入装有10mL的三氯甲烷的烧杯中,超声使其完全溶解, 然后分别加入 6.0 mmol的丙烯酰胺,甲基丙烯酸缩水甘油酯,用滴液漏斗将其滴加到100mL的聚乙烯醇悬浮液中,充分磁力搅拌 2 小时后,再分别加入 6.0 mmol甲基丙烯酸缩水甘油酯、40mmol乙二醇二甲基丙烯酸酯和1.0%的剂偶氮二异丁腈。超声5min,待溶液混合均匀,慢慢滴加到三口瓶中,机械搅拌0,置于75℃恒温油浴中,反应 8h,高速离心得反应产物。
将聚合物放入沸水中重复洗涤6-10次,直至完全除去聚合物表面的聚乙烯醇。然后用甲醇/乙酸 (9∶1,体积/体积)与甲醇清洗反应产物, 直到无模板分子洗出为止。晾干后于40℃下真空干燥48h, 得到表面具有亲水性的分子印迹聚合物。
将分子印迹聚合物放入到50 mL高氯酸水溶液(1:9, V/V)中磁力搅拌24 h,然后抽滤,将聚合物依次在200 mL的乙醇、丙酮、***中不断回流洗脱,最后抽虑,晾干,得到限进介质-分子印迹聚合物。
实施例 3
将1.0 mmol 的6-甲基-4-苯基-2-色满酮加入装有10mL的三氯甲烷的烧杯中,超声使其完全溶解, 然后加入6.0 mmol α-甲基丙烯酸。用滴液漏斗将其滴加到100mL的聚乙烯醇悬浮液中,充分磁力搅拌2 小时后,再分别加入6.0 mmol 甲基丙烯酸缩水甘油酯、60mmol的乙二醇二甲基丙烯酸酯和1.0%的剂偶氮二异丁腈。超声5min,待溶液混合均匀,慢慢滴加到三口瓶中,机械搅拌,置于75℃恒温油浴中,反应 8h,高速离心得反应产物。
将聚合物放入沸水中重复洗涤6-10次,直至完全除去聚合物表面的聚乙烯醇。然后用甲醇/乙酸(9∶1,体积/体积)与甲醇清洗反应产物, 直到无模板分子洗出为止。晾干后于40℃下真空干燥48h, 得到表面具有亲水性的分子印迹聚合物。
将分子印迹聚合物放入到50 mL高氯酸水溶液(1:9, V/V)中磁力搅拌24 h,然后抽滤,将微球依次在200 mL的乙醇、丙酮、***中不断回流洗脱,最后抽虑,晾干,得到限进介质-分子印迹聚合物。
实施例4
研究限进介质-分子印迹材料的吸附性能:将10mg的限进介质-分子印迹聚合物置于5ml离心管中,分别加入4ml不同浓度(0.1、0.2、0.5、1.0、2.0、5.0、8.0、10.0、12.0、15.0μg/mL)的黄曲霉毒素B1的乙腈/水(1:9,V/V)标准溶液,在室温下静置10h。准确移取1mL的上清液,高效液相-荧光检测器测定其吸附后游离黄曲霉毒素的浓度,根据标准曲线计算黄曲霉毒素B1的浓度,根据吸附前后毒素浓度的变化计算聚合物的吸附量Q。同时平行做分子印迹聚合物和限进介质-非印迹聚合物对黄曲霉毒素B1的吸附实验,即将10mg的分子印迹聚合物和限进介质-非印迹聚合物置于5ml离心管中,分别加入4ml黄曲霉毒素B1的乙腈/水标准溶液。随着黄曲霉毒素B1浓度的升高,吸附容量逐渐增大。在相同浓度下,限进介质-分子印迹聚合物的吸附容量大于分子印迹聚合物的吸附容量。
Claims (4)
1.复杂基质中对黄曲霉毒素具有特异性分子识别能力的印迹聚合物的制备方法,其特征在于,包括如下步骤:
(1)黄曲霉毒素替代分子印迹聚合物的合成:在聚乙烯醇溶液中加入替代模板、功能单体、能水解的单体、交联剂、引发剂,加热搅拌反应,结束后用水洗去聚乙烯醇,得到印迹聚合物;所述的功能单体为甲基丙烯酸甲酯,甲基丙烯酸丁酯,丙烯酸,甲基丙烯酸,丙烯酰胺,氯甲基苯乙烯;所述的能水解的单体为甲基丙烯酸缩水甘油酯,丙烯酸缩水甘油酯;所述的交联剂为乙二醇二甲醇丙烯酸酯,二乙烯基苯;所述的引发剂为偶氮二异丁腈,过氧化苯酰;
(2)限进介质-分子印迹聚合物的合成:把(1)制得的印迹聚合物加入到酸的水溶液中水解,反应结束后,抽滤,洗涤,晾干,得到限进介质-分子印迹聚合物;所述酸的水溶液中酸与水的体积比为1:9;
(3)模板分子的洗脱:把(2)制得的聚合物在索氏提取器用有机溶剂-酸的混合物抽提,洗去模板分子,直到不能检测出模板分子为止,再用有机溶剂洗去残留的酸,真空干燥至恒重,得到可以在复杂基质体系中对黄曲霉毒素有选择性吸附的限进介质-印迹聚合物;
步骤(1)中所述的替代模板、功能单体、能水解的单体的摩尔比例为1: 4~9: 3~8;步骤(1)中所述的模板分子和交联剂的摩尔比例为1:10~100。
2.如权利要求1所述的复杂基质中对黄曲霉毒素具有特异性分子识别能力的印迹聚合物的制备方法,其特征在于,步骤(1)中所述的引发剂的加入量为功能单体质量的0.5%~5%,反应温度为50℃~100℃,反应时间为2小时~48小时。
3.如权利要求1所述的复杂基质中对黄曲霉毒素具有特异性分子识别能力的印迹聚合物的制备方法,其特征在于,步骤(2)中,所述的酸为高氯酸。
4.如权利要求1所述的复杂基质中对黄曲霉毒素具有特异性分子识别能力的印迹聚合物的制备方法,其特征在于,步骤(3)中,所述的有机溶剂为甲醇,乙醇,乙腈,所述的酸为乙酸,印迹聚合物用有机溶剂和酸混合液去除模板分子,处理后得到对黄曲霉毒素有选择性吸附的限进介质-印迹聚合物。
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