CN113959968B - 基于AuNPs的比率型比色核酸适配体传感器检测微囊藻毒素MC-LR的方法 - Google Patents
基于AuNPs的比率型比色核酸适配体传感器检测微囊藻毒素MC-LR的方法 Download PDFInfo
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Abstract
本发明公开了一种基于AuNPs的比率型比色核酸适配体传感器检测微囊藻毒素MC‑LR的方法，属于微囊藻毒素检测领域。通过单链适配体的碱基与AuNPs表面的负电荷发生静电作用而直接附着在AuNPs的表面，形成AuNPs/适配体，当MC‑LR存在时，适配体与MC‑LR特异性结合而失去对AuNPs的保护作用，AuNPs进行聚集生长，利用增敏剂NH3OH·HCl和HAuCl4对AuNPs的还原作用，诱导AuNPs的颜色变化，实现MC‑LR的高灵敏检测。本发明方法操作简单、用时短、灵敏方便，不需要依赖大型的设备仪器，对检测环境和时间要求不严格，能够特异性检测MC‑LR，不受其他毒素和离子的干扰。
Description
技术领域
本发明涉及微囊藻毒素检测领域，具体涉及基于盐酸羟胺还原金纳米颗粒的比率型比色核酸适配体传感器快速检测微囊藻毒素-LR的方法。
背景技术
微囊藻毒素-LR(MC-LR)主要通过饮用被污染的水、食用采用污染水源灌溉、蓝藻泥施肥的农作物和蔬菜、食物链传递等途径累积进入生物体内，对其生命安全构成潜在的威胁。MC-LR对动物和人类均会产生毒害效应，不仅对肝器官呈现较强的毒性，还对心、肾、胃肠等器官产生一定的毒害作用。长期暴露在MC-LR中，可能会导致肝癌、心源性心脏病及胃肠炎等疾病，严重的甚至会造成死亡。世界卫生组织(WHO)1998年补充了饮用水质量标准，规定MC-LR(游离态及细胞结合态)的安全阈值为1μg/L。我国在2001年新修订的《生活饮用水卫生规范》将MC-LR列入非常规监测项目，《城市供水水质标准》(CJ/T206-2005)规定MC-LR含量不得超过1μg/L。
传统的检测MC-LR的方法有高效液相色谱法(HPLC)、免疫测定法、液相色谱-质谱联用法(LC-MS)等。虽然这些方法具有高精准度、高灵敏度的特点，但这些技术大多依赖大型设备仪器，在检测环境和时间以及作用成本上当都有较严格的要求。因此，开发一种快速、灵敏、简单、方便的MC-LR检测方法对人体健康、环境监测等方面都具有重要意义。
发明内容
针对现有技术存在的上述问题，本发明所要解决的技术问题在于提供一种基于盐酸羟胺还原金纳米颗粒的比率型比色核酸适配体传感器；本发明所要解决的第二技术问题在于提供一种基于AuNPs的比率型比色核酸适配体传感器检测微囊藻毒素MC-LR的方法。
为了解决上述技术问题，本发明所采用的技术方案如下：
一种AuNPs的比率型比色核酸适配体传感器，通过单链适配体通过碱基与AuNPs表面的负电荷发生静电作用而直接附着在AuNPs的表面，形成AuNPs/适配体，其中，单链适配体的核苷酸序列为：5′-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCGC-3′。
基于所述的AuNPs的比率型比色核酸适配体传感器检测微囊藻毒素MC-LR的方法，包括以下步骤：单链适配体通过碱基与AuNPs表面的负电荷发生静电作用而形成AuNPs/适配体复合体，保护AuNPs发生凝聚。此时加入增敏剂四氯金酸(HAuCl4)和盐酸羟胺(NH3OH·HCl)，因适配体的保护作用，以AuNPs/适配体为核，NH3OH·HCl还原Au3+离子，把AuNPs生长为大小均匀且颜色为红色的AuNPs溶液。当目标物MC-LR存在时，适配体与MC-LR特异性结合而失去对AuNPs的保护作用，AuNPs会发生聚集现象。通过加入增敏剂，聚集的AuNPs为核，生长为尺寸更大且溶液颜色为紫色甚至蓝色的AuNPs。根据反应体系里的AuNPs的颜色变化，可用于可视化检测MC-LR，且通过仪器分析，实现MC-LR的高灵敏检测。
具体步骤如下：
1)将单链适配体、AuNPs、待测样品依次加入到MES缓冲液中，形成反应体系溶液，在37℃反应45min；单链适配体的浓度为50nmol/L，AuNPs在525nm处的吸光度值为0.5且AuNPs的体积占反应体系溶液体积的1/2；
2)在反应体系溶液中加入包含HAuCl4和NH3OH·HCl的增敏剂溶液，建立吸光度比率A650/A525的差值与MC-LR浓度之间的标准曲线，利用绘制的标准曲线测得待测样品中MC-LR的浓度。
进一步的，AuNPs的制备方法为：取0.2mmol/L的HAuCl4溶液，加热至沸腾，沸腾后加入1wt.％柠檬酸三钠溶液，继续加热至溶液变为酒红色时再加热10分钟后停止加热，溶液自然冷却至室温后停止搅拌并用0.45μm的微孔过滤器进行过滤，得到的AuNPs；
本申请中，采用HAuCl4溶液与柠檬酸三钠溶液的体积比为100：7进行制备AuNPs的，可根据实际需要进行相适应的调整。
进一步的，标准曲线的方程为y＝0.0058x+0.0013，MC-LR的检出限为0.3nmol/L。
进一步的，MES缓冲液的浓度为20mmol/L，含1mmol/L Mg2+、3mmol/LK+，pH＝6。
进一步的，反应体系溶液与增敏剂溶液的体积比为1∶1。
进一步的，增敏剂溶液为包含1mmol/L浓度的HAuCl4和2mmol/L浓度的NH3OH·HCl的柠檬酸缓冲溶液；所述柠檬酸缓冲溶液的浓度为10mmol/L，pH＝4.。
相比于现有技术，本发明的有益效果为：
1)本发明构建的比率型比色适配体传感器，利用了核酸适配体的特异性识别实现了对目标MC-LR的快速、简单、灵敏检测；
2)本发明构建的比率型比色适配体传感器，利用增敏剂盐酸羟胺和HAuCl4对AuNPs的还原作用，诱导AuNPs的颜色变化，且通过A650/A525的比率值的计算，最终实现目标MC-LR的高灵敏测定；
3)本发明方法特异性好，具有良好的选择性，受其他毒素和离子受干扰小，如MC-RR、MC-YR、Na+，Ca2+、NH4 +、C1-、NO3 -，CO3 2-，SO4 2-，使用该方法检测MC-LR时，可以排除样品中其他金属离子的干扰，结果更加可靠；
4)本发明方法操作简单，用时短，灵敏方便，不需要依赖大型的设备仪器，对检测环境和时间要求不严格。
附图说明
图1是比率型比色核酸适配体传感器检测MC-LR的工作原理图；
图2是制备的AuNPs的紫外-可见光吸收光谱图；
图3是制备的AuNPs的TEM图；
图4是不同适配体浓度对体系吸光度的影响图；
图5是一系列浓度的MC-LR对体系的AuNPs影响图：图中，(a)为颜色变化图；(b)为紫外-可见吸收光谱图；
图6是MC-LR浓度与吸光度比率差值(ΔA650/A525)的线性关系曲线图；
图7是特异性分析图；图中，MC-LR、MC-RR、MC-YR浓度为50nmol/L，不同离子的浓度均为5μmol/L。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
以下实施例所使用的适配体序列(60mer)：5′-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCGC-3′。
实施例1AuNPs的制备
圆底烧瓶中加入50mL 0.2mmol/L的HAuCl4溶液，并将其放在旋转蒸发仪上，以60rpm、98℃的条件下加热至沸腾。沸腾后加入3.5mL 1％柠檬酸三钠，继续加热至溶液变为酒红色时再加热10分钟后停止加热，溶液自然冷却至室温后停止搅拌并用0.45μm的微孔过滤器进行过滤，得到的溶液即为金纳米颗粒溶液，放置4℃密封保存。
实施例2基于AuNPs的比率型比色核酸适配体传感器用于检测MC-LR的方法
1)适配体浓度的确定
在弱酸性缓冲溶液MES(20mmol/L，pH＝6.0，1mmol/L Mg2+、3mmol/L K+)的环境中，设置0、10、50、100、200nmol/L浓度梯度的MC-LR适配体分别进行对照组和实验组实验(0和1μmol/LMC-LR)。如图4所示，当适配体为50nmol/L时，颜色区分较为明显，且ΔA650/A525值最大，最终选50nmol/L为最佳适配体浓度。
2)MC-LR标准浓度梯度曲线绘制
配制不同浓度的MC-LR溶液，分别加入到含50nmol/L浓度的适配体、一定浓度的AuNPs(AuNPs在525nm处的吸光度值为0.5且AuNPs的体积占反应体系溶液体积的1/2)的20mmol/L MES(含1mmol/L Mg2+、3mmol/L K+)缓冲溶液中，组成反应体系，使反应体系溶液中的MC-LR浓度分别为0、1、5、10、20、50、100、200、500、1000、5000nmol/L，在37℃反应45min。之后，加入与反应体系同体积的增敏剂(含1mmol/LHAuCl4和2mmol/LNHOH·HCl的10mmol/L pH＝4的柠檬酸缓冲溶液)，促进AuNPs的生长。利用酶标仪中的紫外-可见光光谱扫描功能进行测定。如图5所示，随着MC-LR浓度的升高，525nm处的吸光度值降低，而650nm处的吸光度值在升高。根据此现象，利用吸光度比A650/A525的差值(ΔA650/A525)绘制线性关系曲线图(如图6所示)，而且发现MC-LR浓度在0～20nmol/L范围内，ΔA650/A525表现出良好的线性关系，线性回归方程为y＝0.0058x+0.0013，最低检测线为0.3nmol/L(约0.3μg/L)。
3)重金属特异性实验
取50nmol/L的适配体和AuNPs(AuNPs在525nm处的吸光度值为0.5且AuNPs的体积占反应体系溶液体积的1/2)，于20mmol/L MES(含1mmol/LMg2+、3mmol/L K+)缓冲溶液中，以此分别加入不同浓度的微囊藻毒素、阳离子和阴离子(微囊藻毒素-LR(MC-LR)、微囊藻毒素-RR(MC-RR)、微囊藻毒素-YR(MC-YR)、Na+，Ca2+、NH4 +、Cl-、NO3 -，CO3 2-，SO4 2-、Mix)，同时设置超纯水的空白对照，反应体系在37℃反应45min。其中，MC-LR、MC-RR、MC-YR在反应体系中的浓度分别为50nmol/L；其余离子的浓度为5μmol/L；Mix代表把所有的物质混合在一起。之后，加入与反应体系同体积的增敏剂(含1mmol/L HAuCl4和2mmol/L NHOH·HCl的10mmol/LpH＝4的柠檬酸缓冲溶液)，促进AuNPs的生长。利用酶标仪中的紫外-可见光光谱扫描功能进行测定，将各微囊藻毒素、阳、阴离子以及其混合物的ΔA650/A525进行对比。
如图7所示，MC-RR、MC-YR、Na+，Ca2+、NH4 +、Cl-、NO3 -，CO3 2-，SO4 2-等微囊藻毒素和阳、阴离子对体系的ΔA650/A525值几乎没有干扰；而MC-LR和其混合物具有较大的ΔA650/A525值，表明该体系对MC-LR具很好的选择性。
4)实际水样MC-LR的加标实验
选取五种水样类型：河水采样于南京林业大学紫湖溪；湖水采样于玄武湖和洪泽湖；矿泉水选取常见的瓶装饮用水农夫山泉品牌；自来水采样于南京林业大学自来水管道；以及实验室用超纯水。设定50nmol/L为最终加标各水样后测量的MC-LR浓度，测量各水样样品的吸光度，以超纯水作为对照组，重复三组实验。利用酶标仪中的紫外-可见光光谱扫描功能进行测定。选取ΔA650/A525值进行对比，并作误差棒。
如表1所示，此方法对矿泉水、自来水、河水、湖水等5种水样中的MC-LR的加标回收率相当高，均分布在95％～102％之间，其回收率如下：矿泉水样品中回收率为101.66％；自来水样品中回收率为97.51％；河水样品中回收率分别为为99.63％；玄武湖样品中回收率为101.85％；洪泽湖水样品中回收率为95.3％。结果表明，该体系在实际样本中对MC-LR的检测具有准确性和可靠性，可适用于各种环境中的MC-LR定性定量的检测。
表1不同水样的来源与MC-LR的回收率
序列表
<110> 南京林业大学
<120> 基于AuNPs的比率型比色核酸适配体传感器检测微囊藻毒素MC-LR 的方法
<130> 1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 60
<212> DNA
<213> 单链适配体(Artificial)
<400> 1
ggcgccaaac aggaccacca tgacaattac ccataccacc tcattatgcc ccatctccgc 60
Claims (3)
1.基于AuNPs的比率型比色核酸适配体传感器检测微囊藻毒素MC-LR的方法，其特征在于，单链适配体通过碱基与AuNPs表面的负电荷发生静电作用而直接附着在AuNPs的表面，形成AuNPs/适配体，所述单链适配体的核苷酸序列为：5'- GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCGC-3'；
当待测物MC-LR存在时，AuNPs/适配体中的适配体与MC-LR特异性结合而失去对AuNPs的保护作用，AuNPs进行聚集生长，利用增敏剂NH2OH·HCl和HAuCl4对AuNPs的还原作用，增强反应体系里的AuNPs的颜色变化，实现MC-LR的高灵敏检测；
具体包括以下步骤：
1）将单链适配体、AuNPs、待测样品依次加入到MES缓冲液中，形成反应体系溶液，在37℃反应45 min；单链适配体的浓度为50 nmol/L，AuNPs的体积占反应体系溶液体积的1/2且在反应体系溶液中，AuNPs在525 nm处的吸光度值为0.25；MES缓冲液的浓度为20 mmol/L，含1 mmol/L Mg2+、3 mmol/L K+，pH=6；
2）在反应体系溶液中加入包含HAuCl4和NH2OH·HCl的增敏剂溶液，建立吸光度比率A650/A525的差值与MC-LR浓度之间的标准曲线，利用绘制的标准曲线测得待测样品中MC-LR的浓度；所述增敏剂溶液为包含1 mmol/L浓度的HAuCl4和2 mmol/L浓度的NH2OH·HCl的柠檬酸缓冲溶液；所述柠檬酸缓冲溶液的浓度为10 mmol/L，pH=4。
2.根据权利要求1所述的基于AuNPs的比率型比色核酸适配体传感器检测微囊藻毒素MC-LR的方法，其特征在于，AuNPs的制备方法为：取0.2 mmol/L 的HAuCl4溶液，加热至沸腾，沸腾后加入1wt.%柠檬酸三钠溶液，继续加热至溶液变为酒红色时再加热10分钟后停止加热，溶液自然冷却至室温后停止搅拌并用 0.45 μm 的微孔过滤器进行过滤，得到的AuNPs。
3.根据权利要求1所述的基于AuNPs的比率型比色核酸适配体传感器检测微囊藻毒素MC-LR的方法，其特征在于，步骤2）中，反应体系溶液与增敏剂溶液的体积比为1:1。
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