CN107226900B - 一种黄曲霉素b1分子印迹聚合物的制备方法 - Google Patents

一种黄曲霉素b1分子印迹聚合物的制备方法 Download PDF

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CN107226900B
CN107226900B CN201710469461.7A CN201710469461A CN107226900B CN 107226900 B CN107226900 B CN 107226900B CN 201710469461 A CN201710469461 A CN 201710469461A CN 107226900 B CN107226900 B CN 107226900B
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许文革
宋立明
杨琨
陈丽娟
陈元元
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Jilin Province Ainuode Biological Engineering Co Ltd
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Abstract

本发明涉及一种黄曲霉素B1分子印迹聚合物的制备方法，该方法利用单醛对壳聚糖进行预交联；所得的预交联壳聚糖与甲基环氧氯丙烷反应得到壳聚糖环氧树脂预聚物；壳聚糖环氧树脂预聚物与黄曲霉素B1混合后，进行聚合；对包埋了黄曲霉素B1的壳聚糖环氧树脂进行物理、化学清洗，最终得到壳聚糖环氧树脂。本发明利用亲水性壳聚糖作为聚合单体，首先用单醛封闭壳聚糖上的氨基反应位点，保证了壳聚糖环氧树脂预聚物的顺利合成；用单醛封闭壳聚糖上的氨基反应位点，保证了壳聚糖环氧树脂与黄曲霉素B1不会发生共价结合；单醛上的烷基链为黄曲霉素B1中的疏水基团提供了完美的疏水环境，使得壳聚糖环氧树脂预聚物与黄曲霉素B1具有良好的亲附性。

Description

一种黄曲霉素B1分子印迹聚合物的制备方法
技术领域
本发明涉及一种分子印迹聚合物的制备方法，特别是一种黄曲霉素B1分子印迹聚合物的制备方法。
背景技术
黄曲霉毒素(AFT)在1993年就已经被世界卫生组织(WHO)的癌症研究机构划定为I类致癌物。在天然污染的食品和饲料中以黄曲霉毒素B1(AFB1)最为常见，其毒性和致癌性也最强，不仅给社会带来重大的经济损失，而且严重威胁消费者的健康。世界各国及地区均制定了严格的AFT限量标准，且限量要求日益严格。目前黄曲霉毒素的检测方法有薄层层析法、高效液相色谱法、酶联免疫吸附法、放射免疫测定法、免疫亲和层析柱-高效液相色谱法、免疫亲和层析柱- 荧光光度法等。目前免疫亲和层析柱-高效液相色谱法和免疫亲和层析柱-荧光光度法，具有快速、灵敏、准确的特点，已被国家标准GB/T18979-2003所采用。然而，免疫亲和柱所需的抗体制备不易，且质量稳定性与产品稳定性都难以控制。
分子印迹聚合物是一类以小分子化合物为模板，通过物理包埋和化学聚合而制备的新型功能材料。分子印迹的制备理念与抗原抗体的亲和原理非常接近。由于分子印迹聚合物可选择的聚合单体具有化学多样性、能够充分预测最终聚合物的结构和功能属性，该聚合物通常都选择结构上没有活泼基团的单体，因此具有热稳定性好和使用寿命长等优点。由于其聚合过程中包埋了分子印迹，而聚合物的化学稳定性导致分子印迹能够非常稳定的保持下去。因此，分子印迹聚合物可以作为高灵敏度的物理吸附剂广泛应用于生物、药品和食品等样品处理及检测等方面。
壳聚糖作为一种廉价易得的生物高分子材料已经被应用于分子印迹的研究之中。壳聚糖分子中具有氨基和羟基，因此改性的方式多，反应灵活。然而，如何利用壳聚糖上的活性基团，制备出特异性的分子印迹是一个研究难点。
发明内容
本发明的目的是提供一种黄曲霉素B1分子印迹聚合物的制备方法，为现有黄曲霉素B1检测方法提供了全新的思路，利用针对黄曲霉素B1的分子印迹聚合物作为吸附剂，对样品中的黄曲霉素B1的含量进行检测，具有操作方便，聚合物可做到无限次回收套用，对于黄曲霉素B1的检测灵敏度高，检测结果准确等优点。
为实现上述目的，本发明提供的黄曲霉素B1分子印迹聚合物的制备方法，利用单醛对壳聚糖进行预交联，所得的预交联壳聚糖与甲基环氧氯丙烷反应得到壳聚糖环氧树脂预聚物；壳聚糖环氧树脂预聚物与黄曲霉素B1混合后，进行聚合；然后通过对包埋了黄曲霉素B1的壳聚糖环氧树脂进行物理、化学清洗，最终得到壳聚糖环氧树脂，用壳聚糖环氧树脂即可快速检测出黄曲霉素B1。
本发明利用亲水性壳聚糖作为聚合单体，首先用单醛封闭壳聚糖上的氨基反应位点，能够完美的控制壳聚糖中氨基的活性，从而使与甲基环氧氯丙烷的反应位点控制在壳聚糖上的羟基位置，同时也保证了壳聚糖环氧树脂与黄曲霉素B1不会发生共价结合；单醛上的烷基链以及环氧基的聚合反应发生后产生的结合态氧为黄曲霉素B1提供了良好的极性与非极性环境。
本发明提供的一种黄曲霉素B1分子印迹聚合物的制备方法，具体的制备步骤如下：
1)壳聚糖预交联步骤如下：
将一定量的壳聚糖溶于乙酸水溶液，置于装有搅拌器及温度计的500mL三口烧瓶中；加入液体石蜡，启动搅拌器，常温下搅拌10min后升温到50℃，滴加适量的乳化剂，乳化10min，然后升温到60℃，滴加一定量的单醛，搅拌反应 3h；冷却，加入水溶性溶剂帮助沉淀，过滤，滤饼用不溶于水的溶剂洗涤三次，将过滤得到的滤饼于60℃真空干燥，得到壳聚糖预交联产物。
其中，所述的壳聚糖交联所用的乳化剂为span60、吐温80、卵磷脂、大豆磷脂中的一种或几种，优选为span60、吐温80或卵磷脂；所述的壳聚糖交联所用的单醛为碳链长度1-5的单醛，优选为甲醛、乙醛或丙醛；所述水溶性溶剂为碳链长1-5的短链醇、丙酮，优选为甲醇、乙醇或丙酮；所述的不溶于水的溶剂为乙醚、沸点30-60℃的石油醚、沸点60-90℃的石油醚、正己烷、环己烷，优选为石油醚、正己烷或环己烷。
2)壳聚糖环氧树脂预聚物步骤如下：
将步骤1)得到的壳聚糖预交联产物分散于适量活化剂中，升温到90℃，反应4h，反应完毕，利用旋转蒸发仪将反应体系的溶剂蒸干，将蒸干剩余物溶于不溶于水的溶剂中，以恒压滴液漏斗缓慢滴加质量分数为48.5％的氢氧化钠水溶液，反应4h，反应完毕用水洗至中性，得到壳聚糖环氧树脂预聚物混合液。
其中，所述的活化剂为甲基环氧氯丙烷；所述的不溶于水的溶剂为正己烷、环己烷、苯、甲苯、乙苯，优选为甲苯、乙苯或正己烷。
3)黄曲霉素B1分子印迹聚合物步骤如下：
取黄曲霉素B1加入步骤2)得到的壳聚糖环氧树脂预聚物混合液中，0℃条件下以恒压滴液漏斗缓慢滴加固化剂，反应2h，将反应液分散于不溶于水的溶剂中，得到的固体沉降，过滤，用溶于水的溶剂洗涤三次，干燥，最终得到黄曲霉素B1分子印迹聚合物。
其中，所述的固化剂为乙二胺、己二胺、二乙烯三胺、三乙烯四胺、二乙氨基丙胺、顺丁烯二酸酐、邻苯二甲酸酐，三氟化硼乙醚溶液(CAS:109-63-7，市售)；优选为三氟化硼乙醚溶液、己二胺或顺丁烯二酸酐；所述的不溶于水的溶剂为二氯甲烷、二氯乙烷、氯仿、乙酸乙酯、正己烷、环己烷，优选为二氯甲烷、正己烷或二氯乙烷；所述的溶于水的溶剂为碳链长1-5的短链醇、丙酮，优选为甲醇、乙醇或丙醇。
本发明的优点和有益效果在于：
(1)本发明提供的黄曲霉素B1分子印迹聚合物的制备方法，利用亲水性壳聚糖作为聚合单体。a)首先用单醛封闭壳聚糖上的氨基反应位点，保证了壳聚糖环氧树脂预聚物的顺利合成；b)用单醛封闭壳聚糖上的氨基反应位点，保证了壳聚糖环氧树脂与黄曲霉素B1不会发生共价结合；c)单醛上的烷基链为黄曲霉素B1中的疏水基团提供了完美的疏水环境，使得壳聚糖环氧树脂预聚物与黄曲霉素B1具有良好的亲附性；d)环氧基的聚合反应发生后产生的结合态氧为黄曲霉素B1提供了良好的极性环境。
(2)采用本发明提供的方法制备黄曲霉素B1分子印迹聚合物，可快速检测出黄曲霉素B1，且黄曲霉素B1的回收率高达98.5％，该分子印迹化合物使用寿命长，经过脱附洗涤可重复利用。
附图说明
图1为实施例1壳聚糖的预交联化学反应图。
图2为实施例1壳聚糖环氧树脂预聚物化学反应图。
图3为黄曲霉素B1分子印迹聚合物化学反应示意图。
图4为壳聚糖、实施例1、实施例2、实施例3化合物的红外谱图，其中A 为壳聚糖、B为实施例1的分子印迹聚合物、C为实施例2的分子印迹聚合物、 D为实施例3的分子印迹聚合物。
具体实施方式
为使本领域技术人员清楚明白本发明的技术方案，下面通过以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。
实施例1
1)壳聚糖的预交联：
将2.02g壳聚糖溶于200ml2％乙酸水溶液，置于装有搅拌器及温度计的 500mL三口烧瓶中。加入200ml液体石蜡，启动搅拌器，常温下搅拌10min后升温到50℃，滴加3mlspan60，乳化10min，然后升温到60℃，滴加5.8g的甲醛，搅拌反应3h。冷却，加入200ml丙酮帮助沉淀，过滤，滤饼用100ml石油醚洗涤三次，将过滤得到的滤饼于60℃真空干燥，得到壳聚糖预交联产物。
2)壳聚糖环氧树脂预聚物的制备：
将步骤1)得到的壳聚糖预交联产物分散于30ml甲基环氧氯丙烷中，升温到90℃，反应4h，反应完毕，利用旋转蒸发仪将反应体系的溶剂蒸干，将蒸干剩余物溶于50ml甲苯，以恒压滴液漏斗缓慢滴加2.8g质量分数为48.5％的氢氧化钠水溶液，反应4h，反应完毕用水洗至中性，得到壳聚糖环氧树脂预聚物混合液。
3)黄曲霉素B1分子印迹聚合物的制备：
取1.8g黄曲霉素B1加入步骤2)得到的壳聚糖环氧树脂预聚物混合液中。 0℃条件下以恒压滴液漏斗缓慢滴加1.5ml三氟化硼/乙醚溶液，反应2h，将反应液分散于100ml二氯甲烷中，得到的固体沉降，过滤，用100ml乙醇洗涤三次，干燥，最终得到黄曲霉素B1分子印迹聚合物。
实施例2
1)壳聚糖的预交联：
将2.02g壳聚糖溶于200ml2％乙酸水溶液，置于装有搅拌器及温度计的500mL三口烧瓶中。加入200ml液体石蜡，启动搅拌器，常温下搅拌10min后升温到50℃，滴加3ml吐温80，乳化10min，然后升温到60℃，滴加7.5g的乙醛，搅拌反应3h。冷却，加入200ml甲醇帮助沉淀，过滤，滤饼用100ml正己烷洗涤三次，将过滤得到的滤饼于60℃真空干燥，得到壳聚糖预交联产物。
2)壳聚糖环氧树脂预聚物的制备：
将步骤1)得到的壳聚糖预交联产物分散于30ml甲基环氧氯丙烷中，升温到90℃，反应4h，反应完毕，利用旋转蒸发仪将反应体系的溶剂蒸干，将蒸干剩余物溶于50ml乙苯，以恒压滴液漏斗缓慢滴加2.9g质量分数为48.5％的氢氧化钠水溶液，反应4h，反应完毕用水洗至中性，得到壳聚糖环氧树脂预聚物混合液。
3)黄曲霉素B1分子印迹聚合物的制备：
取1.8g黄曲霉素B1加入步骤2)得到的壳聚糖环氧树脂预聚物混合液中。 0℃条件下以恒压滴液漏斗缓慢滴加1.5ml乙二胺，反应2h，将反应液分散于 100ml正己烷中，得到的固体沉降，过滤，用100ml甲醇洗涤三次，干燥，最终得到黄曲霉素B1分子印迹聚合物。
实施例3
1)壳聚糖的预交联：
将2.02g壳聚糖溶于200ml2％乙酸水溶液，置于装有搅拌器及温度计的 500mL三口烧瓶中。加入200ml液体石蜡，启动搅拌器，常温下搅拌10min后升温到50℃，加入3.5g卵磷脂，乳化10min，然后升温到60℃，滴加9.4g的丙醛，搅拌反应3h。冷却，加入200ml乙醇帮助沉淀，过滤，滤饼用100ml环己烷洗涤三次，将过滤得到的滤饼于60℃真空干燥，得到壳聚糖预交联产物。
2)壳聚糖环氧树脂预聚物的制备：
将步骤1)得到的壳聚糖预交联产物分散于30ml甲基环氧氯丙烷中，升温到90℃，反应4h，反应完毕，利用旋转蒸发仪将反应体系的溶剂蒸干，将蒸干剩余物溶于50ml正己烷，以恒压滴液漏斗缓慢滴加3.1g质量分数为48.5％的氢氧化钠水溶液，反应4h，反应完毕用水洗至中性，得到壳聚糖环氧树脂预聚物混合液。
3)黄曲霉素B1分子印迹聚合物的制备：
取1.8g黄曲霉素B1加入步骤2)得到的壳聚糖环氧树脂预聚物混合液中。 0℃条件下以恒压滴液漏斗缓慢滴加1.7ml顺丁烯二酸酐，反应2h，将反应液分散于100ml二氯乙烷中，得到的固体沉降，过滤，用100ml丙醇洗涤三次，干燥，最终得到黄曲霉素B1分子印迹聚合物。
实施例4分子印迹聚合物中黄曲霉素B1负载量计算
取上述实施例制备的三种微球，分别加入到黄曲霉素B1的甲醇溶液中，在摇床中振荡吸附，在吸附时间内分时间点取上清液测定含量。待吸附饱和，测定溶液中黄曲霉素B1的浓度变化，利用下列公式来计算静态吸附量。实施例1至实施例3分子印迹聚合物静态吸附数据见表1。
Q0＝(Co-Cx)*V0/Mx
Q0：静态吸附量(mg/g)；
V0：黄曲霉素B1溶液体积(ml)；
Co：黄曲霉素B1溶液的初始浓度(mg/ml)；
Cx：吸附平衡后黄曲霉素B1溶液的浓度(mg/ml)；
Mx：吸附平衡时微球的用量(g)。
实施例5分子印迹聚合物中黄曲霉素B1的回收率计算
取吸附饱和后三种分子印迹聚合物先经布氏漏斗抽滤，然后在漏斗上方缓慢加入少量二次水洗涤，然后将微球放入索氏提取器中，以甲醇作为萃取剂对微球中吸附的物质进行循环萃取，分时间点取萃取液测定含量，直至萃取液的浓度不发生变化，利用下式确定黄曲霉素B1的回收率。回收率数据见表1。
Uy＝(Cx*V1/Q0*Mx)*100％
Uy：静态脱附率(％)；
Cx：回收完成后溶液中黄曲霉素B1的浓度(mg/ml)；
V1：回收溶剂总体积(ml)；
Mx：吸附平衡时微球的用量(g)。
表1：分子印迹聚合物对黄曲霉素B1的吸附量以及回收率数据
参阅图4，从未改性的壳聚糖的红外光谱可以看到，3200cm－1～3500cm－1处具有一个较大的宽峰，从归属上来看，应为O-H的伸缩振动吸收峰存在的区间以及N-H的伸缩振动吸收峰存在的区间，因此考虑是两种伸缩振动吸收峰重叠而成。而1000cm－1附近的吸收峰则可以归属为壳聚糖分子中所具有的羟基中的C-O伸展与O-H面内变形振动吸收峰的这两个峰叠加到一起的结果。而 1601cm－1处的吸收峰则是N-H的弯曲振动吸收带所提供的。
而三种分子印迹聚合物的红外光谱表明：1)氨基的振动吸收峰(3358cm－1处的小分叉)消失表明其发生了化学反应；2)氨基的弯曲振动吸收带消失，也证明了这一点；3)饱和C-H伸缩振动的吸收峰2924cm－1和2869cm－1比未改性的壳聚糖明显增强，这说明希夫碱的形成使得壳聚糖上引入了碳链。

Claims (8)

1.一种黄曲霉素B1分子印迹聚合物的制备方法，其特征在于：该方法包括以下步骤：
1)壳聚糖的预交联：
将壳聚糖溶于乙酸水溶液，置于装有搅拌器及温度计的500mL三口烧瓶中；加入液体石蜡，启动搅拌器，常温下搅拌10min后升温到50℃，滴加适量的乳化剂，乳化10min，然后升温到60℃，滴加单醛，搅拌反应3h；冷却，加入水溶性溶剂帮助沉淀，过滤，滤饼用不溶于水的溶剂洗涤三次，将过滤得到的滤饼于60℃真空干燥，得到壳聚糖预交联产物；
2)壳聚糖环氧树脂预聚物的制备：
将步骤1)得到的壳聚糖预交联产物分散于适量活化剂中，升温到90℃，反应4h，反应完毕，利用旋转蒸发仪将反应体系的溶剂蒸干，将蒸干剩余物溶于不溶于水的溶剂中，以恒压滴液漏斗缓慢滴加质量分数为48.5％的氢氧化钠水溶液，反应4h，反应完毕用水洗至中性，得到壳聚糖环氧树脂预聚物混合液；所述的活化剂为甲基环氧氯丙烷；
3)黄曲霉素B1分子印迹聚合物的制备：
取黄曲霉素B1加入步骤2)得到的壳聚糖环氧树脂预聚物混合液中，0℃条件下以恒压滴液漏斗缓慢滴加固化剂，反应2h，将反应液分散于不溶于水的溶剂中，得到的固体沉降，过滤，用溶于水的溶剂洗涤三次，干燥，最终得到黄曲霉素B1分子印迹聚合物。
2.根据权利要求1所述的一种黄曲霉素B1分子印迹聚合物的制备方法，其特征在于：步骤1)中所述的乳化剂为span60、吐温80、卵磷脂、大豆磷脂中的一种或几种；所述的单醛为碳链长1-5的单醛；所述水溶性溶剂为碳链长1-5的短链醇、丙酮；所述的不溶于水的溶剂为乙醚、沸点30-60℃的石油醚、沸点60-90℃的石油醚、正己烷、环己烷。
3.根据权利要求1所述的一种黄曲霉素B1分子印迹聚合物的制备方法，其特征在于：步骤2)中所述的不溶于水的溶剂为正己烷、环己烷、苯、甲苯、乙苯。
4.根据权利要求1所述的一种黄曲霉素B1分子印迹聚合物的制备方法，其特征在于：步骤3)中所述的固化剂为乙二胺、己二胺、二乙烯三胺、三乙烯四胺、二乙氨基丙胺，顺丁烯二酸酐、邻苯二甲酸酐，三氟化硼乙醚溶液；所述的不溶于水的溶剂为二氯甲烷、氯仿、乙酸乙酯、正己烷、环己烷；所述的溶于水的溶剂为碳链长1-5的短链醇、丙酮。
5.根据权利要求2所述的一种黄曲霉素B1分子印迹聚合物的制备方法，其特征在于：步骤1)中所述的乳化剂为span60、吐温80或卵磷脂；所述的单醛为甲醛、乙醛或丙醛；所述水溶性溶剂为甲醇、乙醇或丙酮；所述的不溶于水的溶剂为石油醚、正己烷或环己烷。
6.根据权利要求3所述的一种黄曲霉素B1分子印迹聚合物的制备方法，其特征在于：步骤2)中所述的不溶于水的溶剂为甲苯、乙苯或正己烷。
7.根据权利要求4所述的一种黄曲霉素B1分子印迹聚合物的制备方法，其特征在于：步骤3)中所述的固化剂为三氟化硼乙醚溶液、己二胺或顺丁烯二酸酐；所述的不溶于水的溶剂为二氯甲烷、正己烷；所述的溶于水的溶剂为甲醇、乙醇或丙醇。
8.根据权利要求1所述的一种黄曲霉素B1分子印迹聚合物的制备方法，其特征在于：步骤1)中壳聚糖与单醛质量比为1：2-5。
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