CN109507126A - 一种水产品中孔雀石绿检测方法 - Google Patents
一种水产品中孔雀石绿检测方法 Download PDFInfo
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- G—PHYSICS
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- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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Abstract
本发明公开了一种水产品中孔雀石绿检测方法，它包括磁性分子印迹聚合物的制备和基于核酸适配体和四氧化三铁模拟酶催化方法的建立等实验步骤。本发明首先用多巴胺聚合法制备孔雀石绿(MG)磁性分子印迹聚合物(MG‑MMIPs)，利用MG‑MMIPs对样品中的微量MG进行吸附，达到富集目标物和去除复杂介质的目的，再将富集的MG解吸后，利用核酸适配体和四氧化三铁模拟酶催化的检测法进行检测。该方法的灵敏度为5nmol L‑1，最低检出限达到3.6nmol L‑1。对MG的两种结构类似物(灿烂绿和结晶紫)以及其他抑菌药(磺胺胍、磺胺、盐酸土霉素和氯霉素)的特异性实验表明，该方法对MG检测具有良好的选择性。
Description
技术领域
本发明涉及一种较大范围，具体是一种水产品中孔雀石绿检测方法。
背景技术
孔雀石绿(Malachite green，MG)是一种有毒的三苯甲烷类物质，又称为盐基块绿、碱性孔雀绿、孔雀绿、碱性绿、中国绿或苯胺绿，为具有金属光泽的深绿色结晶状固体。孔雀石绿也可用作杀菌剂，对于防止霉菌在鱼卵中生长以及真菌的二次污染有较好的效果。但是，孔雀石绿存在高毒、高残留和高致癌、诱变的缺点。但由于其价格低廉，而且杀菌效果显著，且没有合适的替代品，目前仍有不法商户在使用。由于其添加量很少就可以达到很好的杀菌效果，给检测水产品中的MG带来一定的难度。因此，建立MG残留的检测方法十分迫切并且具有重要的现实意义。
目前MG的检测方法主要有高效液相色谱法、液相色谱-质谱联用法、气相色谱-质谱联用法以及酶联免疫法。这几种方法在测定和分析水产品中孔雀石绿的残留都具有结果可靠、灵敏度高、选择性高、重复性好的优点。但是这些方法在样品前处理过程都存在繁琐、复杂、耗时等不足，而且设备昂贵，检测成本高；不仅需要消耗大量的溶剂和大量的时间，还容易造成二次污染，影响检测结果的准确性；酶联免疫法则需要制备抗体，操作复杂，且易受pH值、温度等环境条件的影响，检测条件苛刻。因此，发展一种简单高效的MG检测方法对控制水产品质量安全具有重要意义。
磁性分子印迹聚合物具有构效稳定、富集效率高、特异性强和适用性广等特点，能够对MG起到富集的作用，提高了检测的灵敏度，而基于核酸适配体和四氧化三铁模拟酶催化的检测法具有操作简单、快速、经济的特点。将二者结合起来，以合成的磁性分子印迹聚合物富集MG后用四氧化三铁模拟酶催化检测体系进行检测，不仅提高了 MG检测灵敏度，还提高了体系的选择性。
发明内容
本发明的目的在于提供一种水产品中孔雀石绿检测方法，以解决上述背景技术中提出的问题。
为实现上述目的，本发明提供如下技术方案：
一种水产品中孔雀石绿检测方法，所述水产品为基于磁性分子印迹聚合物和核酸适配体的四氧化三铁模拟酶催化的水产品，具体步骤如下：
(1)Fe3O4的合成：称取0.30～0.36g聚乙二醇，加入7～9mL 超纯水，超声直至聚乙二醇完全溶解，称取2.00～2.06g FeCl3加入到聚乙二醇溶液中超声溶解，再称取1.4～1.6g FeCl2加入到上述溶液中至完全溶解，立即加入0.4～0.6mL 0.1mol L-1稀盐酸，通氮除氧12～18min，60℃水浴下边搅拌边迅速加入55～65mL氨水，调转速至1500rmin-1，反应1h，用磁铁进行吸附，并用超纯水洗脱4～6次，真空冷冻干燥后保存备用；
(2)MG磁性分子印迹聚合物的制备：称取100～200mg Fe3O4加到30ml的Tris-HCl缓冲液20mmol L-1,pH 8.5中，再加入6mg MG 和90mg盐酸多巴胺混匀，即MG浓度为0.2g L-1，多巴胺浓度为3g L-1。在电动搅拌器下反应4h，用磁铁进行吸附，并用甲醇：乙酸为 9:1(v/v)混合液洗脱至无MG模板洗出，再用甲醇洗至中性，最后用超纯水洗去甲醇，烘干后备用。
(3)Fe3O4模拟酶催化剂的制备：称取1.30～1.40g FeCl3加到 30～50mL乙二醇中搅拌溶解，再加入3.4～3.8g无水乙酸钠和800～ 1000μg PEG-200搅拌30min后转移到反应釜中，200℃下反应12h。冷却至室温后将反应物转移出反应釜，用超纯水和乙醇反复清洗，真空干燥备用。
(4)检测：称取10～20mg磁性分子印迹聚合物于10mL离心管中，加入3mL浓度为5～100nmol L-1的MG水溶液，吸附5min 后用磁铁分离，弃去上清液。加入1mL甲醇乙酸(9:1，v/v)混合液洗脱15min，重复3次，合并提取液。用氮气吹干提取液，立即加入300μL醋酸钠缓冲液200mmol L-1,pH 4.0，充分溶解后备用。在 0.5ml离心管中加入10μL Fe3O4模拟酶催化剂和30μL 2.5μmol L-1 MG DNA核酸适配体，充分震荡混合30min后分别加入50μL一系列浓度的MG提取液，充分混合30min，最后加入80μL 1μmol L-1 TMB3,3',5,5'-四甲基联苯胺溶液和80μL 1mol L-1H2O2溶液，震荡10 min后用磁铁将Fe3O4模拟酶催化剂分离，上清液转入酶标板中，用酶标仪读取650nm处的吸光度值。
作为本发明进一步的方案：所述步骤(2)中聚合物用多巴胺。
作为本发明进一步的方案：所述步骤(3)中模拟酶催化剂为 Fe3O4。
作为本发明进一步的方案：所述步骤(4)中所用核酸适配体为 DNA。
作为本发明进一步的方案：所述步骤(4)中显色剂为TMB。
与现有技术相比，本发明的有益效果是：本发明磁性分子印迹聚合物具有特异识别性和富集性以及分离杂质等显著特点，而基于核酸适配体和四氧化三铁模拟酶催化的检测法所用的核酸适配体DNA也具有良好的特异识别能力，且具有简单、快速和灵敏的特点，将二者结合起来，用磁性分子印迹聚合物富集低浓度的检测物后，再用基于核酸适配体和四氧化三铁模拟酶催化的检测体系进行检测，提高了检测灵敏度和选择性。
附图说明
图1为本发明中磁性分子印迹聚合物吸附动力学图；
图2为本发明中磁性分子印迹聚合物等温吸附曲线图；
图3本发明中Fe3O4模拟酶催化剂透射电镜图；
图4为本发明中Fe3O4模拟酶催化剂磁滞回归曲线；
图5为本发明中基于磁性分子印迹聚合物和核酸适配体的四氧化三铁模拟酶催化的水产品中孔雀石绿检测方法检测MG的紫外-可见吸收光谱图；
图6为本发明中基于磁性分子印迹聚合物和核酸适配体的四氧化三铁模拟酶催化的水产品中孔雀石绿检测方法检测MG的标准曲线；
图7为本发明中基于磁性分子印迹聚合物和核酸适配体的四氧化三铁模拟酶催化的水产品中孔雀石绿检测方法的选择性图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明实施例
一种水产品中孔雀石绿检测方法，所述水产品为基于磁性分子印迹聚合物和核酸适配体的四氧化三铁模拟酶催化的水产品，具体步骤如下：
(1)Fe3O4的合成：称取0.33g聚乙二醇，加入8mL超纯水，超声直至聚乙二醇完全溶解，称取2.03g FeCl3加入到聚乙二醇溶液中超声溶解，再称取1.5g FeCl2加入到上述溶液中至完全溶解，立即加入0.5mL 0.1mol L-1稀盐酸，通氮除氧15min，60℃水浴下边搅拌边迅速加入60mL氨水，调转速至1500rmin-1，反应1h，用磁铁进行吸附，并用超纯水洗脱5次，真空冷冻干燥后保存备用；
(2)MG磁性分子印迹聚合物的制备：称取200mg Fe3O4加到30ml的Tris-HCl缓冲液20mmol L-1,pH 8.5中，再加入6mg MG和 90mg盐酸多巴胺混匀，即MG浓度为0.2g L-1，多巴胺浓度为3g L-1。在电动搅拌器下反应4h，用磁铁进行吸附，并用甲醇：乙酸为9:1 (v/v)混合液洗脱至无MG模板洗出，再用甲醇洗至中性，最后用超纯水洗去甲醇，烘干后备用。
(3)Fe3O4模拟酶催化剂的制备：称取1.35g FeCl3加到40mL 乙二醇中搅拌溶解，再加入3.6g无水乙酸钠和900μg PEG-200搅拌 30min后转移到反应釜中，200℃下反应12h。冷却至室温后将反应物转移出反应釜，用超纯水和乙醇反复清洗，真空干燥备用。
(4)检测：称取15mg磁性分子印迹聚合物于10mL离心管中，加入3mL浓度为5-100nmol L-1的MG水溶液，吸附5min后用磁铁分离，弃去上清液。加入1mL甲醇乙酸(9:1，v/v)混合液洗脱15min，重复3次，合并提取液。用氮气吹干提取液，立即加入300μL醋酸钠缓冲液200mmol L-1,pH 4.0，充分溶解后备用。在0.5ml离心管中加入10μLFe3O4模拟酶催化剂和30μL 2.5μmol L-1MG DNA核酸适配体，充分震荡混合30min后分别加入50μL一系列浓度的MG提取液，充分混合30min，最后加入80μL 1μmol L-1TMB3,3',5,5'-四甲基联苯胺溶液和80μL1mol L-1H2O2溶液，震荡10min后用磁铁将 Fe3O4模拟酶催化剂分离，上清液转入酶标板中用酶标仪读取650nm 处的吸光度值。
绘制650nm处吸光度随MG变化的标准曲线如图6所示。
优选的所述步骤(2)中Tris-HCl缓冲液pH值为8.5.
优选的所述步骤(2)中多巴胺浓度为3g L-1。
优选的所述步骤(4)中DNA浓度为2.5μmol L-1；TMB浓度为 1μmol L-1；H2O2浓度为1mol L-1。
由图1可知，制备的磁性分子印迹MIP和磁性非印迹NIP有明显的吸附差异，且在5min时达到吸附平衡。
由图2可知，制备的MIP在MG浓度为80mg L-1时达到最大吸附量44.5μg g-1，而NIP在MG浓度为80mg L-1时，达到最大吸附量约为18.7μg g-1。
由图3可知，制备的Fe3O4模拟酶催化剂为近球形结构，粒径大约为200nm。
由图4可知，制备的Fe3O4模拟酶催化剂具有很好的超顺磁性，饱和磁性强度为71.3emu g-1。由此可知制备出的Fe3O4在外加磁场作用下可以迅速从反应体系中分离出来。
由图5和图6可知，所建立的基于磁性分子印迹聚合物和核酸适配体的四氧化三铁模拟酶催化的水产品中孔雀石绿检测体系，随着 MG浓度的增加，650nm处吸光度出现逐渐上升的趋势，且与MG 浓度呈现良好的线性关系，R2达到0.991.
所建立的基于磁性分子印迹聚合物和核酸适配体的四氧化三铁模拟酶催化的水产品中孔雀石绿检测方法的特异性验证：选择孔雀石绿及其结构类似物灿烂绿(BG)、结晶紫(CV)以及其他抑菌剂磺胺胍(SG)、磺胺(SAs)、盐酸土霉素(OTH)、氯霉素(CAP)作为特异性分析物，由图7可以看出，在650nm处含有MG的体系吸光度值较高，而含有其他物质的吸光度值较低，结果表明MG加入体系后有响应，而其他分析物响应较弱，可以看出该检测体系选择性良好。
实际样品的添加回收实验：在养殖水样和鱼样中分别加入10、 25、50nmol L-1孔雀石绿标准溶液，然后采用建立的基于磁性分子印迹聚合物和核酸适配体的四氧化三铁模拟酶催化的水产品中孔雀石绿检测方法测定样品回收率，如表2所示实验结果表明，所建立的方法能够应用于实际样品检测中，并且检测效果较好。
表1本研究所用MG核酸适配体序列
表2基于磁性分子印迹和核酸适配体的四氧化三铁模拟酶催化体系对样品中孔雀石绿检测的加标回收实验
Claims (5)
1.一种水产品中孔雀石绿检测方法，所述水产品为基于磁性分子印迹聚合物和核酸适配体的四氧化三铁模拟酶催化的水产品，具体步骤如下：
(1)Fe3O4的合成：称取0.30～0.36g聚乙二醇，加入7～9mL超纯水，超声直至聚乙二醇完全溶解，称取2.00～2.06g FeCl3加入到聚乙二醇溶液中超声溶解，再称取1.4～1.6gFeCl2加入到上述溶液中至完全溶解，立即加入0.4～0.6mL 0.1mol L-1稀盐酸，通氮除氧12～18min，60℃水浴下边搅拌边迅速加入55～65mL氨水，调转速至1500r min-1，反应1h，用磁铁进行吸附，并用超纯水洗脱4～6次，真空冷冻干燥后保存备用；
(2)MG磁性分子印迹聚合物的制备：称取100～200mg Fe3O4加到30ml的Tris-HCl缓冲液20mmol L-1,pH 8.5中，再加入6mg MG和90mg盐酸多巴胺混匀，即MG浓度为0.2g L-1，多巴胺浓度为3g L-1。，在电动搅拌器下反应4h，用磁铁进行吸附，并用甲醇：乙酸为9:1(v/v)混合液洗脱至无MG模板洗出，再用甲醇洗至中性，最后用超纯水洗去甲醇，烘干后备用。；
(3)Fe3O4模拟酶催化剂的制备：称取1.30～1.40g FeCl3加到30～50mL乙二醇中搅拌溶解，再加入3.4～3.8g无水乙酸钠和800～1000μg PEG-200搅拌30min后转移到反应釜中，200℃下反应12h。冷却至室温后将反应物转移出反应釜，用超纯水和乙醇反复清洗，真空干燥备用。；
(4)检测：称取10～20mg磁性分子印迹聚合物于10mL离心管中，加入3mL浓度为5～100nmol L-1的MG水溶液，吸附5min后用磁铁分离，弃去上清液，加入1mL甲醇乙酸(9:1，v/v)混合液洗脱15min，重复3次，合并提取液。用氮气吹干提取液，立即加入300μL醋酸钠缓冲液200mmol L-1,pH 4.0，充分溶解后备用，在0.5ml离心管中加入10μL Fe3O4模拟酶催化剂和30μL 2.5μmol L-1MG DNA核酸适配体，充分震荡混合30min后分别加入50μL一系列浓度的MG提取液，充分混合30min，最后加入80μL 1μmol L-1TMB3,3',5,5'-四甲基联苯胺溶液和80μL 1mol L-1H2O2溶液，震荡10min后用磁铁将Fe3O4模拟酶催化剂分离，上清液转入酶标板中，用酶标仪读取650nm处的吸光度值。
2.根据权利要求1所述的一种水产品中孔雀石绿检测方法，其特征在于，所述步骤(2)中聚合物用多巴胺。
3.根据权利要求1所述的一种水产品中孔雀石绿检测方法，其特征在于，所述步骤(3)中模拟酶催化剂为Fe3O4。
4.根据权利要求1所述的一种水产品中孔雀石绿检测方法，其特征在于，所述步骤(4)中所用核酸适配体为DNA。
5.根据权利要求1所述的一种水产品中孔雀石绿检测方法，其特征在于，所述步骤(4)中显色剂为TMB。
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