CN108195828A - 一种非标记均相检测苯甲酸钠的比色法 - Google Patents
一种非标记均相检测苯甲酸钠的比色法 Download PDFInfo
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Abstract
一种非标记均相检测苯甲酸钠的比色法。本发明利用聚联乙炔囊泡受到外界扰动由蓝变红的光学特性，将联乙炔单体与苯甲酸半抗原——对氨基苯甲酸相连，制成囊泡探针，光聚合后囊泡显蓝色。该囊泡体系与苯甲酸抗体特异性结合，囊泡显红色，当检测体系中存在游离苯甲酸，囊泡探针由蓝变红的程度收到干扰，降低的程度与苯甲酸含量相关，由此建立了苯甲酸钠的快速检测体系。该法无需特殊仪器，定性测定时仅凭肉眼可视，定量测定时采用常规紫外分光光度计即可；操作简单，非均相测试，无需酶标记；方法灵敏，检测线性范围为0.01ng mL‑1‑100ng mL‑1，食品样品处理仅稀释即可，具有简单、经济、选择性好、灵敏度高等优点。
Description
技术领域
本发明属于分析检测技术领域，具体涉及一种非标记型均相比色检测苯甲酸钠的方法。
背景技术
苯甲酸(C7H6O2)又称安息香酸，是苯环上的一个氢被羧基(-COOH)取代形成的化合物。在酸性条件下对真菌、细菌、霉菌的生长和繁殖有很强的抑制作用，故在食品中常作为防腐剂使用。苯甲酸分子态的抑菌活性较离子态高，故pH＜4时，抑菌活性高，抑菌的最适pH值为2.5～4.0，一般以低于pH值4.5～5.0为宜。由于苯甲酸对水的溶解度低，故实际多是加适量的碳酸钠或碳酸氢钠，用90℃以上热水溶解，使其转化成苯甲酸钠后才添加到食品中。但由于其对人体有一定毒性，特别是过量使用时会对人体造成危害，我国《食品安全国家标准-食品添加剂使用标准(GB-2760-2011)》限定了苯甲酸及其钠盐的使用范围和使用量。近年来，食品安全事件接连发生，食品安全问题引起了人们的高度关注。食品添加剂在食品中的残留检测有十分重要的意义。目前食品中苯甲酸钠的测定方法主要有气相色谱法/高效液相色谱法、薄层色谱法，这些方法测定准确度高，但操作相对复杂，需要专门高价仪器设备。因此，急需研究一种简便、经济、高灵敏和高选择性的苯甲酸检测方法。
发明内容
针对现有技术的不足，本发明要解决的问题是提供一种能快速检测苯甲酸钠的检测方法。聚联乙炔囊泡由于其特殊的光学性质受到广泛关注，由于自身是两亲分子，联乙炔会以囊泡的形式存在于溶液中，紫外光照射下联乙炔分子中两个相邻炔基通过1，4-加成反应生成聚联乙炔，可见光激发其分子骨架离域电子，使其产生π-π*跃迁，溶液显示为蓝色，紫外-可见光谱中最大吸收约为650nm。外界环境变化，如发生抗原-抗体反应等因素影响到囊泡骨架构象，离域电子运动状态也发生变动，紫外-可见光谱中最大吸收峰蓝移到540nm，囊泡体系转变为红色。
依据聚联乙炔囊泡由蓝色转变为红色的程度，可用比色响应值(ColorimetricResponse，CR)定量表示：CR(％)＝(PB0-PBf/PB0)×100％式中：PB＝Ablue/(Ablue+Ared)，Ablue和Ared分别为囊泡体系蓝色(紫外吸收波长650nm)或红色(紫外吸收波长540nm)状态的吸收值；PB0为空白对照组囊泡体系Ablue/(Ablue+Ared)的比值；PBf为对环境响应后发生后囊泡体系Ablue/(Ablue+Ared)的值。由公式可知，囊泡颜色未发生改变时CR％＝0％，CR％值越大表明体系颜色转变程度越强，反之则越小。基于该性质，将联乙炔单体与苯甲酸半抗原-对氨基苯甲酸相连，制成囊泡探针，光聚合后囊泡显蓝色，该囊泡体系与苯甲酸抗体特异性结合，囊泡显红色，当检测体系中存在游离苯甲酸，囊泡探针由蓝变红的程度收到干扰，CR％值降低，降低的程度与苯甲酸含量相关，由此建立苯甲酸钠的快速检测体系。该发明的优势在于：(1)测试无需特殊仪器。定性测定时仅凭肉眼可视，定量测定时采用常规紫外分光光度计即可；(2)操作简单，非均相测试，无需酶标记和分离；(3)方法灵敏，且食品样品处理简单。液体样品无需萃取、分离等操作，稀释即可，稀释100倍仍能满足测试要求。
1.本发明所采用的技术方案由下述步骤组成：
(1)制备蓝色囊泡溶液
取1mM PCDA的二氯甲烷储液与1mM PCDA-对氨基苯甲酸储液按照一定比例体积比混合后加1mM DMPC储液，于100mL圆底烧瓶减压旋干后加入超纯水，80℃超声15min，室温放置2h。转移至4℃存放12h后以紫外254nm激发15min，得到蓝色囊泡溶液。
(2)制作标准曲线
向300μL蓝色囊泡溶液中加入10μL OVA(卵清蛋白)封闭液，10μL苯甲酸钠标准溶液(浓度为0.01ng mL-1、0.1ng mL-1、1ng mL-1、10ng mL-1、100ng mL-1、1000ng mL-1、10000ngmL-1)，之后加入10μL苯甲酸钠抗体，涡旋振荡混匀，37℃孵育30min，用紫外分光光度计测定上述不同浓度苯甲酸钠标准溶液的紫外吸收光谱，计算CR％值，以浓度为0.01-10000ngmL-1苯甲酸钠溶液为横坐标，比色响应值CR％值为纵坐标，由origin8.0软件作图得标准曲线。
作为优选，步骤(1)中所述中的PCDA单体与DMPC的比例以7∶3最优，得到比较灵敏的检测囊泡探针体系。
作为优选，步骤(2)中所述制作标准曲线，选取的标准溶液的浓度为0.01ng mL-1、0.1ng mL-1、1ng mL-1、10ng mL-1、100ng mL-1、1000ng mL-1、10000ng mL-1。
作为优选，步骤(2)中所述制作标准曲线，孵育的时间选定为30min。
附图说明：
图1 PDA囊泡探针体系中加入不同浓度苯甲酸钠的紫外光谱图
图2 PDA囊泡探针快速测定苯甲酸钠的标准曲线图
图3苯甲酸钠HPLC法的测定标准曲线
具体实施方式：
(1)制备囊泡
取1mM PCDA的二氯甲烷储液与1mM PCDA-对氨基苯甲酸储液按照7∶3(23mL∶10mL)的体积比混合，之后加1mM DMPC储液14mL，在100mL圆底烧瓶减压旋干后加入47mL超纯水，80℃超声15min，之后室温放置2小时，转移至4℃放置12h，紫外254nm激发15min，得到蓝色囊泡溶液。
(2)制作标准曲线
向300μL囊泡溶液中加入10μL OVA封闭液，10μL苯甲酸钠标准溶液(浓度为0.01ngmL-1、0.1ng mL-1、1ng mL-1、10ng mL-1、100ng mL-1、1000ng mL-1、10000ng mL-1)，之后加入10μL苯甲酸钠抗体。涡旋振荡混匀，37℃孵育30min，用紫外分光光度计测定上述不同浓度苯甲酸钠标准溶液的紫外吸收光谱，以浓度为0.01-10000ng mL-1苯甲酸钠溶液为横坐标，比色响应值CR％为纵坐标，由origin8.0软件作图得标准曲线。
(3)检测样品溶液中的苯甲酸钠
①样品的预处理：
量取样品(酱油、醋)1mL，超纯水稀释100倍，备用。
②向300μL囊泡溶液中加入10μL OVA封闭液，10μL样品溶液，涡旋混匀后加入10μL苯甲酸钠抗体，用紫外分光光度计测定上述不同浓度苯甲酸钠标准溶液的紫外光谱，计算CR％，代入标准曲线求得样品中苯甲酸钠含量。
同时依据文献建立HPLC法，测定样品中的苯甲酸钠含量，用以对比，结果见表1。
表1.本发明方法和HPLC法检测醋和酱油中的苯甲酸含量
Claims (5)
1.一种非标记均相检测苯甲酸钠的比色法，由下述步骤组成：
(1)制备蓝色囊泡溶液
取1mM PCDA的二氯甲烷储液与1mM PCDA-对氨基苯甲酸储液按照一定比例体积比混合后加1mM DMPC储液，于100mL圆底烧瓶减压旋干后加入超纯水，80℃超声15min，室温放置2h，转移至4℃存放12h后以紫外254nm激发15min，得到蓝色囊泡溶液；
(2)制作标准曲线
向300μL蓝色囊泡溶液中加入10μL OVA(卵清蛋白)封闭液，10μL苯甲酸钠标准溶液(浓度为0.01ng mL-1、0.1ng mL-1、1ng mL-1、10ng mL-1、100ng mL-1、1000ng mL-1、10000ng mL-1)，之后加入10μL苯甲酸钠抗体，涡旋振荡混匀，37℃孵育30min，用紫外分光光度计测定上述不同浓度苯甲酸钠标准溶液的紫外吸收光谱，计算CR％值，以浓度为0.01-10000ng mL-1苯甲酸钠溶液为横坐标，比色响应值CR％值为纵坐标，由origin8.0软件作图得标准曲线；
(3)检测样品溶液中的苯甲酸钠
①样品的预处理：
样品(酱油/醋)，超纯水稀释100倍，备用；
②同上操作：向300μL蓝色囊泡溶液中加入10μL OVA封闭液，10μL样品溶液，涡旋混匀后加入10μL苯甲酸钠抗体，用紫外分光光度计测定上述不同浓度苯甲酸钠标准溶液的紫外光谱，计算CR％值，代入标准曲线求得样品中苯甲酸钠含量。
2.按照权利要求1所述的非标记型均相比色检测苯甲酸钠的方法，其特征在于所述使用PCDA-对氨基苯甲酸作为构建囊泡的关键单体成分，PCDA一种常用的联乙炔单体，为10，12-二十五二炔酸，DMPC为二肉豆蔻酰磷脂酰胆碱，用作制备囊泡的磷脂成分，囊泡探针的特征在于：在制备囊泡时，聚联二炔与磷脂的比例为7∶3，旋蒸去除溶剂后，加入等体积蒸馏水，超声分散，用紫外分光光度计测定紫外光谱。
3.按照权利要求1所述的非标记型均相比色检测苯甲酸钠的方法，其特征在于：在制备囊泡时，PCDA与PCDA-对氨基苯甲酸按照一定比例混合，优选6∶4，7∶3，8∶2，9∶1，优选7∶3。
4.根据权利要求1所述的非标记型均相比色检测苯甲酸钠的方法，其特征在于：检测测定样品的紫外吸收强度，计算CR％值，与标准工作曲线相比，计算待检测溶液中苯甲酸钠的含量。
5.根据权利要求1所述的苯甲酸钠的检测方法，其特征在于：所述检测液的吸收波长是蓝色(650nm)或红色(540nm)。
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