CN106018398A - 一种快速定性筛查铅离子的方法 - Google Patents
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Abstract
本发明公开了一种快速定性筛查铅离子的方法，经过样品预处理、初筛反应场所的选择、初筛试剂的配置、初筛反应和定性分析步骤，完成对铅离子的筛查；本发明基于铅离子能显著抑制G4/hemin复合物过氧化物酶性，而其他金属离子不能明显抑制其过氧化物酶性特点，操作简便可行，方法重现性良好，能快速筛查出食品安全、环境监测等领域中的重金属铅，能满足日常大批量样品快速筛查铅离子要求。
Description
技术领域
本发明属于食品、环境检测领域，具体涉及一种快速定性筛查铅离子的方法。
背景技术
重金属通常指密度大于5g/cm3以上的金属如金、银、铜、铅、锌、镍、钴、镉、铬和汞等45种金属。重金属污染泛指铅、汞、镉、砷等重金属的污染，通过生物链来污染人类赖以生存的动植物产品及水源。重金属进入人体后，主要危害的是抑制酶活性，破坏正常的酶的代谢。
重金属常用检测方法包括比色法、试纸法、酶学法、电化学法、原子吸收法、原子荧光光谱法、电感耦合等离子体法等。比色法、试纸法操作设备简单、操作简便，但是方法的灵敏度和选择性不高；电化学法、原子吸收法、原子荧光光谱法、电感耦合等离子体法等方法具有较高的灵敏度，往往需要大型仪器和较强的专业人才；酶学法具有特异性强、灵敏度高的优点，能运用于大批样品的检测，但也有酶的成本较高和生物活性不稳定等局限性。
重复的富含G碱基的结构单元的DNA序列能形成堆积的G4链体结构。生物信息学研究表明超过376,000个潜在的G4链体结构单元在人的基因组中，至少40%的人的人的基因启动子含有1个或几个G4结构单元。G4链体结构PS2.M与氯化血红素形成复合物后具有较高的过氧化物酶活性。笔者曾报道了G4与氯化血红素形成的G4/氯化血红素复合物具有过氧化物酶普适性，并探讨了其活性与结构的相关性。
发明内容
本发明的目的在于针对现有技术的不足，现提供一种使用方便、
检测效果佳的快速定性筛查铅离子的方法。
为解决上述技术问题，本发明采用的技术方案为：一种快速定性筛查铅离子的方法，其创新点在于：经过样品预处理、初筛反应场所的选择、初筛试剂的配置、初筛反应和定性分析步骤，完成对铅离子的筛查；具体步骤如下：
（1）样品预处理：复杂的食品需经过强酸微波消解、电热板赶酸、高速离心和滤纸过滤处理，处理后的食品冷却后用缓冲液定量转移至10mL容量瓶中，定容至刻度，混匀后待测；环境样品需添加0.1-1g的活性炭吸附悬浮物和色素，滤纸过滤，调节pH值6.5-7.5，混匀后待测；
（2）初筛反应场所的选择：酶标板、小试管或小离心管；
（3）初筛试剂的配置：10mmol/L氯化血红素储备液完成DMSO配置，-20℃保存备用，所用G-四链体G4 DNA、氯化血红素、过氧化物酶底物、H2O2和缓冲液均需现用现配；所用G4 DNA用缓冲液溶解，并在95oC环境下加热5min，冰上冷却至室温，在4oC条件下静置过夜；
（4）初筛反应：将10µL 20µmol/L的G4 DNA、10µL 10µmol/L的氯化血红素和20µL1mmol/L金属离子室温作用0.5h，然后加入80µL 5mmol/L过氧化物酶底物和80µL 5mmol/LH2O2，混合均匀；各物质终浓度分别为：G4 DNA浓度为1µmol/L，氯化血红素浓度为0.5µmol/L，金属离子浓度为0.1mmol/L，过氧化物酶底物浓度为2mmol/L，H2O2浓度为2mmol/L；
（5）定性分析：用酶标仪监测铅离子抑制RET或HIF-1α与氯化血红素形成复合物过氧化反应30秒内产生的自由基阴离子的动力学，记录414nm吸光度随时间的变化，每两秒钟采集一次吸光度信号，若待测样品较空白样品抑制动力学存在显著差异，可初步判断可能存在铅离子；反应5分钟后，肉眼目视比较待测样品与空白样品反应液颜色的深浅，若待测样品较空白样品存在颜色显著差异，可初步判断可能存在铅离子。
进一步的，所述缓冲液包括20mmol/L pH 7.4的Tris/HCl、100mmol/L的 NaCl、10mmol/L的KCl、0.01%浓度的Triton X-100和1%浓度的DMSO。
进一步的，所述步骤（5）中的RET为RET原癌基因，序列为：5'-GGG CGG GCG CGGGCG GG-3’；所述HIF-1α为缺氧诱导因子1α基因启动子，序列为：5'-GGG AGG GAG AGG GGGCGGG-3’。
本发明的有益效果如下：本发明基于铅离子能显著抑制G4/hemin 复合物过氧化物酶性，而其他金属离子不能明显抑制其过氧化物酶性特点，操作简便可行，方法重现性良好，能快速筛查出食品安全、环境监测等领域中的重金属铅，能满足日常大批量样品快速筛查铅离子要求。
附图说明
图1 为常见金属离子对过氧化物酶活性的抑制能力的RET/氯化血红素复合物体系图；
图2为茶叶样品中铅离子初筛的RET/氯化血红素复合物体系图；
图3为常见金属离子对过氧化物酶活性的抑制能力的HIF-1α/氯化血红素复合物体系图；
图4为水样中铅离子初筛的HIF-1α/氯化血红素复合物体系图。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式，熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效。
RET为RET原癌基因，序列为：5'-GGG CGG GCG CGG GCG GG-3’；HIF-1α为缺氧诱导因子1α基因启动子，序列为：5'-GGG AGG GAG AGG GGG CGGG-3’。
实施例1
RET/氯化血红素复合物体系：准确称取0.1986g茶叶样品1和0.2004g茶叶样品2，茶叶样品2中加入1mL 1mol/L的醋酸铅，将茶叶粉碎均匀后置于50mL微波消解罐中，加入2mL硝酸、1mL过氧化氢，盖好安全阀，放入微波炉消解系统消解，消解完全后，电热板上赶去残余硝酸，冷却后用缓冲液定量转移至10mL容量瓶中定容至刻度，混匀备用，同时做试剂空白试验。
10µL 20µmol/LRET G4, 10µL 10µmol/L氯化血红素，20µL茶叶样品1和茶叶样品2置于室温作用0.5h，然后加入80µL 5mmol/L ABTS，80µL 5mmol/L H2O2，混合均匀。
如图1和图2所示，未加醋酸铅的茶叶样品1，几乎保持了原来和RET/氯化血红素复合物过氧化物酶活性，反应5分钟后体系颜色较深；而加入醋酸铅的茶叶样品2，较强地抑制了RET/氯化血红素复合物过氧化物酶活性，反应5分钟后体系颜色较浅。
实施例2
HIF-1α/氯化血红素复合物体系：准确量取18.0 mL水样两份，记为水样3和水样4，水样4中加入2 mL 1.0mol/L醋酸铅，加入0.1g活性炭，混匀，10分钟后，过滤除活性炭；调节两份水样pH 7.0左右后待测；
10µL 20µmol/L HIF-1αG4, 10µL 10µmol/L氯化血红素，20µL水样3和水样4室温作用0.5h，加入80µL 5mmol/L ABTS，80µL 5mmol/L H2O2；
如图3和图4所示，未加醋酸铅的水样3，几乎保持了原来和HIF-1α/氯化血红素复合物过氧化物酶活性，反应5分钟后体系颜色较深；而加入醋酸铅的水样4，较强地抑制了HIF-1α/氯化血红素复合物过氧化物酶活性，反应5分钟后体系颜色较浅。
本发明基于铅离子能显著抑制G4/hemin 复合物过氧化物酶性，而其他金属离子不能明显抑制其过氧化物酶性特点，操作简便可行，方法重现性良好，能快速筛查出食品安全、环境监测等领域中的重金属铅，能满足日常大批量样品快速筛查铅离子要求。
上述实施例只是本发明的较佳实施例，并不是对本发明技术方案的限制，只要是不经过创造性劳动即可在上述实施例的基础上实现的技术方案，均应视为落入本发明专利的权利保护范围内。
Claims (3)
1.一种快速定性筛查铅离子的方法，其特征在于：经过样品预处理、初筛反应场所的选择、初筛试剂的配置、初筛反应和定性分析步骤，完成对铅离子的筛查；具体步骤如下：
（1）样品预处理：复杂的食品需经过强酸微波消解、电热板赶酸、高速离心和滤纸过滤处理，处理后的食品冷却后用缓冲液定量转移至10mL容量瓶中，定容至刻度，混匀后待测；环境样品需添加0.1-1g的活性炭吸附悬浮物和色素，滤纸过滤，调节pH值6.5-7.5，混匀后待测；
（2）初筛反应场所的选择：酶标板、小试管或小离心管；
（3）初筛试剂的配置：10mmol/L氯化血红素储备液完成DMSO配置，-20℃保存备用，所用G-四链体G4 DNA、氯化血红素、过氧化物酶底物、H2O2和缓冲液均需现用现配；所用G4 DNA用缓冲液溶解，并在95oC环境下加热5min，冰上冷却至室温，在4oC条件下静置过夜；
（4）初筛反应：将10µL 20µmol/L的G4 DNA、10µL 10µmol/L的氯化血红素和20µL1mmol/L金属离子室温作用0.5h，然后加入80µL 5mmol/L过氧化物酶底物和80µL 5mmol/LH2O2，混合均匀；各物质终浓度分别为：G4 DNA浓度为1µmol/L，氯化血红素浓度为0.5µmol/L，金属离子浓度为0.1mmol/L，过氧化物酶底物浓度为2mmol/L，H2O2浓度为2mmol/L；
（5）定性分析：用酶标仪监测铅离子抑制RET或HIF-1α与氯化血红素形成复合物过氧化反应30秒内产生的自由基阴离子的动力学，记录414nm吸光度随时间的变化，每两秒钟采集一次吸光度信号，若待测样品较空白样品抑制动力学存在显著差异，可初步判断可能存在铅离子；反应5分钟后，肉眼目视比较待测样品与空白样品反应液颜色的深浅，若待测样品较空白样品存在颜色显著差异，可初步判断可能存在铅离子。
2.根据权利要求1所述的一种快速定性筛查铅离子的方法，其特征在于：所述缓冲液包括20mmol/L pH 7.4的Tris/HCl、100mmol/L的 NaCl、10mmol/L的KCl、0.01%浓度的TritonX-100和1%浓度的DMSO。
3.根据权利要求1所述的一种快速定性筛查铅离子的方法，其特征在于：所述步骤（5）中的RET为RET原癌基因，序列为：5'-GGG CGG GCG CGG GCG GG-3’；所述HIF-1α为缺氧诱导因子1α基因启动子，序列为：5'-GGG AGG GAG AGG GGG CGGG-3’。
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