CN114878559A - 一种裸眼检测中药材和食品中玉米赤霉烯酮的方法 - Google Patents
一种裸眼检测中药材和食品中玉米赤霉烯酮的方法 Download PDFInfo
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Abstract
本发明提供一种裸眼检测中药材和食品中玉米赤霉烯酮(zearalenone，ZEN)的方法，涉及中药材、食品安全检测技术领域。该裸眼检测中药材和食品中玉米赤霉烯酮的方法，包括如下步骤：将4μL Buffer,5μL Na,5μL K和2.5μL DNAzyme混匀，形成溶液A，向溶液A加入含有ZEN的溶液，混匀后避光孵育30min，加入10μL溶液B，溶液B为ABTS和H2O2混合溶液，利用酶标仪记录溶液在472nm处的吸收值，用智能手机拍照记录每一分钟溶液颜色变化情况。通过Photoshop软件对实时照片进行RGB通道转化后观察图片颜色变化来实现ZEN的裸眼可视化检测，优点在于操作简单，易携带，低成本，不需要专业的技术人员，稳定性好，高灵敏性，可作为用于现场分析中药材及食品样品中ZEN的常用工具。
Description
技术领域
本发明涉及中药材、食品安全检测技术领域，具体为一种裸眼检测中药材和食品中玉米赤霉烯酮的方法。
背景技术
玉米赤霉烯酮(ZEN)又称F-2毒素，由镰刀菌代谢产生，是世界上分布最广的镰刀菌毒素之一。ZEN及其衍生物主要存在于发霉的中药材(川贝母、当归、苦杏仁等)、谷类(玉米、大米、小麦、大麦、燕麦、高粱等)、乳制品中。ZEN具有雌激素作用，短期大量摄入可引起急性中毒，出现恶心，呕吐，腹泻等症状；长期接触则易导致慢性中毒(>40毫克/公斤)。ZEN具有较强的致突变性，致畸性，致癌性，神经毒性和生殖毒性，易导致患上如肝癌，子宫纤维化，乳腺囊肿，垂体癌以及宫颈癌等疾病。此外，ZEN还具有生产快，残留时间长，耐高温，难以去除等特点，因此，亟待开发一种可以快速现场检测ZEN的方法用于监控中药材或食品质量。
目前，色谱法常用于样品中ZEN浓度的检测，如气相色谱-质谱(GC-MS)、高效液相色谱(HPLC)、高效液相色谱-质谱(HPLC-MS)等。这些方法具有高灵敏度和高特异性的优点，但它们耗时且昂贵，需要专业的分析人员和设备，样品预处理繁琐及分析方法复杂。除色谱法外，以酶联免疫吸附试验(ELISA)为代表的免疫法通过巧妙的设计虽可实现裸眼可视化检测，但其依赖于特定的抗体，成本高，不同批次的抗体存在较大的差异，且运输和保存条件较为苛刻，严重限制了免疫法在现场即时检测中的应用。
发明内容
针对现有技术的不足，本发明的目的是提供一种裸眼现场检测中药材和食品中玉米赤霉烯酮的方法，该方法具有不需要修饰，操作简单，成本低，稳定，便携，可裸眼实时监测，不需要专业技术人员操作等优点。
为实现以上目的，本发明通过以下技术方案予以实现：一种裸眼检测中药材和食品中玉米赤霉烯酮的方法，包括以下步骤：
步骤一：将Buffer,Na,K和DNAzyme混匀，形成溶液A；
步骤二：向溶液A中加入含有ZEN的溶液，混匀后避光孵育30min；
步骤三：再加入含ABTS和H2O2的混合溶液B；
步骤四：利用酶标仪记录溶液的吸收值，拍照记录溶液颜色变化情况。
优选的，所述步骤一中，溶液A中的原料配比为：4μL Buffer、5μL Na、5μL K、2.5μLDNA探针。
优选的，所述DNAzyme的序列为：5’-GGGXGGGXXGGGXGGG-3’，其中X为A、T、C三种碱基中的任意一个。
优选的，所述步骤二中，混匀后避光孵育30min。
优选的，所述步骤三中，加入的ABTS和H2O2混合溶液为10μL。
优选的，所述步骤四具体包括：利用酶标仪记录溶液在472nm处的吸收值，用智能手机拍照记录每一分钟溶液颜色变化情况。
本发明的检测原理：DNAzyme与血红素(Hemin)结合成为复G-quadruplex，当体系中不存在ZEN时，DNAzyme在H2O2的作用下将ABTS催化成绿色的ABTS·+；当存在ZEN时，体系生成一种新的物质，其在472nm处有最大吸收峰，溶液呈黄色，通过对实时照片进行G→RGB转化后观察图片颜色变化来实现ZEN的POCT检测。
本发明提供了一种裸眼检测中药材和食品中玉米赤霉烯酮的方法。具备以下有益效果：
本发明通过将Buffer,Na,K和DNAzyme混匀，形成溶液A，向溶液A中加入ZEN，混匀后避光孵育30min，再加入ABTS和H2O2混合溶液，利用酶标仪记录溶液的吸收值，拍照记录溶液颜色变化情况，优点在于操作简单，易携带，低成本，不需要专业的技术人员，稳定性好，高灵敏性，可作为用于现场分析中药材食品样品中ZEN的常用工具。
附图说明
图1为本发明的方法原理图；
图2为本发明实施例中DNAzyme用于ZEN检测示意图；
图3为本发明实施例中DNAzyme检测不同浓度ZEN的示意图；
图4为本发明实施例中DNAzyme用于检测川贝母，当归，苦杏仁，大米中的ZEN示意图(其主要是通过像这些样品中添加已知浓度的ZEN制得检测样品)。
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。
实施例中所涉及的工艺方法，如无特殊说明则为常规方法或步骤，所用药品试剂除特殊说明外，均为市售。本发明所涉及的术语，除非另外定义，否则本文所用的所有技术及科学术语都具有与本发明所属领域的普通技术人员通常所了解相同的含义。
实例1:DNAzyme用于ZEN的检测
如图1-2所示，本发明实施例提供一种裸眼检测中药材和食品中玉米赤霉烯酮的方法，包括如下步骤：
第一步：将4μL Buffer,5μL Na,5μL K和2.5μL DNAzyme混匀，形成溶液A。
第二步：向溶液A加入含有ZEN的溶液，混匀后避光孵育30min。
第三步：加入10μL ABTS和H2O2混合溶液，ABTS和H2O2混合溶液为溶液B。
第四步：利用酶标仪记录溶液在472nm处的吸收值，用智能手机拍照记录每一分钟溶液颜色变化情况。结果如图2，DNAzyme用于ZEN的检测。其中ZEN(-)为不加ZEN，ZEN(+)为加入100μM的ZEN。插入中的照片为30min时两个样品溶液的颜色。
DNA模拟酶(DNAzyme)与血红素(Hemin)结合后，具有模拟HRP的催化活性，在H2O2的作用下将无色的2,2'-叠氮双(3-乙基苯并噻唑-6-磺酸)(ABTS)催化氧化成绿色的ABTS·+。研究者前期发现，在ZEN的存在下，ABTS不能被催化氧化成绿色的ABTS·+，而是生成新的产物，该产物的最大波长为472nm，溶液颜色变为黄色。当不存在ZEN时，DNAzyme可将ABTS催化成ABTS·+，溶液呈深绿色。基于这一发现，本发明利用DNAzyme构建了一种能够快速、裸眼可视化检测中药材及食品中的ZEN的新方法。本发明有望实现ZEN的现场即时筛查，为保证药品、食品等产品的安全奠定一定的实验基础，并拓展ZEN检测传感器的设计思路。
实例2:ZEN浓度依赖曲线的建立
采用实例1中的检测步骤，利用DNAzyme进行检测。
其中ZEN待测溶液浓度分别为0，0.05，0.5，1，5，10，20，50，80，100，200，300μM。
记录472nm处的吸收值，并进行拟合，并对样品拍照，最后将15min时拍的图片进行G→RGB转化。
DNAzyme检测结果如图3。图3为DNAzyme检测不同浓度的ZEN示意，其中，(a)为DNAzyme对不同浓度的ZEN的信号响应，(b)为对0-300μM的ZEN的标准曲线。
实例3:DNAyme对大米，川贝，当归，苦杏仁中加入已知浓度的ZEN并检测。
采用实例1中的检测步骤，其中ZEN溶液为100μM，记录472nm处的吸收值。结果如图4。图4为DNAzyme检测川贝母，当归，苦杏仁，大米中的ZEN示意。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
Claims (6)
1.一种裸眼检测中药材和食品中玉米赤霉烯酮的方法，其特征在于：包括以下步骤：
步骤一：将Buffer,Na,K和DNAzyme混匀，形成溶液A；
步骤二：向溶液A中加入ZEN，混匀后避光孵育；
步骤三：再加入含ABTS和H2O2的混合溶液B；
步骤四：利用酶标仪记录溶液的吸收值，拍照记录溶液颜色变化情况。
2.根据权利要求1所述的一种裸眼检测中药材和食品中玉米赤霉烯酮的方法，其特征在于：所述步骤一中，溶液A中的原料配比为：4μL Buffer、5μL Na、5μL K、2.5μL DNA探针。
3.根据权利要求1所述的一种裸眼检测中药材和食品中玉米赤霉烯酮的方法，其特征在于：所述DNAzyme的序列为：5’-GGGXGGGXXGGGXGGG-3’，其中X为A、T、C三种碱基中的任意一个。
4.根据权利要求1所述的一种裸眼检测中药材和食品中玉米赤霉烯酮的方法，其特征在于：所述步骤二中，混匀后避光孵育30min。
5.根据权利要求1所述的一种裸眼检测中药材和食品中玉米赤霉烯酮的方法，其特征在于：所述步骤三中，加入的ABTS和H2O2混合溶液B为10μL。
6.根据权利要求1所述的一种裸眼检测中药材和食品中玉米赤霉烯酮的方法，其特征在于：所述步骤四具体包括：利用酶标仪记录溶液在472nm处的吸收值，用智能手机拍照记录每一分钟溶液颜色变化情况。
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