CN114858791B - 一种裸眼可视化检测硫化氢的方法 - Google Patents
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Abstract
本发明提供一种裸眼可视化检测硫化氢的方法，涉及工业、环境、医疗、生活安全检测技术领域。该方法，主要包括以下步骤：将含有10‑40mM HEPES缓冲溶液、200‑400mM NaCl、50‑100mM KCl、0.5‑2μM AgNO3、10‑100mM罗丹明B、1μM DNA1、1μM DNA2混合，向上述溶液中加入0‑500μM H2S5μL；反应后加入Triton‑X‑100和Hemin共3μL，反应15‑30min；反应后加入10μL含有10‑40mM HEPES缓冲溶液、2mM H2O2、1.5mM ABTS的溶液至总体积为50μL，并利用酶标仪记录1‑30min时溶液在418nm吸收值，拍照记录溶液颜色变化情况。本发明利用RGB通道进行颜色转化，避免主观因素导致的差异，具有快速、便捷、裸眼可视的特点，有望实现硫化氢的现场快速检测，为工业、环境、医疗、生活中硫化氢检测带来便捷。
Description
技术领域
本发明涉及工业、环境、医疗、生活安全检测技术领域，具体为一种裸眼可视化检测硫化氢的方法。
背景技术
硫化氢(H2S)标准状况下是一种酸性气体，无色，低浓度时有臭鸡蛋气味，为一种剧毒的空气污染物。其广泛存在于如硫酸、硫磺、染料、化妆品等工业生产过程，以及厌氧菌还原无机硫酸盐或有机硫化物的微生物降解过程。据报道，人体吸入500-1000ppm(partsper million)会导致死亡。1996年美国学者Abe和Kimura提出H2S是继NO和CO的第三种内源性产生的气态化合物，普遍存在于脑、肝脏、肾脏和心血管系统中，并参与各种生理过程，如血管生成、血管扩张、细胞凋亡、调节炎症和神经调节等。细胞中的H2S水平异常与众多疾病有关，如阿尔茨海默病、唐氏综合症、糖尿病和肝硬化等。由此可见，开发一种高效的方法来检测环境及生命系统中的H2S对于预防H2S中毒和了解其生理、病理功能具有重要的意义。
目前常用的H2S检测方法有：比色分析法、电化学分析法、色谱法和荧光探针法等。其中电化学分析法对环境变化敏感、易受碱性环境及杂质干扰、需要校准；色谱法需要特殊装备，检测比较耗时；荧光探针法是近年来随着荧光传感技术的应用被认为是研究生物H2S的很有前途的工具，但荧光探针的制备往往需要费力的程序和昂贵的反应物和试剂，无法达到方便快捷的检测目的；比色分析法因其成本低、操作简单，被认为是一种理想的方法。更重要的是，比色分析法可通过肉眼进行实时观察，且不需要任何复杂的仪器，但目前可用于H2S的裸眼检测方法极少，依赖于酶联免疫分析的比色检测费时且成本高，极大的限制了其在临床的应用。因此，亟需开发一种安全稳定、方便快捷的裸眼可视化检测硫化氢的方法。
发明内容
针对现有技术的不足，本发明的目的是提供一种快速、便捷、安全、稳定的硫化氢检测方法，此方法对样本需求量小、检测试剂安全稳定、操作简单、结果肉眼可见，非专业人员也可进行检测。
为实现以上目的，本发明通过以下技术方案予以实现：一种裸眼可视化检测硫化氢的方法，包括以下步骤：
步骤一、将含有10-40mM HEPES缓冲溶液、200-400mM NaCl、50-100mM KCl、0.5-2μM AgNO3、10-100mM罗丹明B、1μM DNA1、1μM DNA2混合，用灭菌后的UP水定容至32μL；
步骤二、向上述溶液中加入0-500μM H2S 5μL，反应15-30min；
步骤三、反应后加入Triton-X-100和Hemin共3μL，反应15-30min；
步骤四、反应后加入10μL含有10-40mM HEPES缓冲溶液、2mM H2O2、1.5mM ABTS的溶液至总体积为50μL，并利用酶标仪记录1-30min时溶液在418nm吸收值，用手机拍照记录溶液颜色变化情况并进行RGB转化。
优选的，DNA1的序列为：5’-AAA AAA ACT CTC TCT CTC TCT CTC TCT CAT TATTTT AGG GCG GG-3’。
优选的，DNA2的序列为：5’-GGG CGG GAA AAA TAA TCA CAC ACA CAC ACA CACACA CTT TTT TT-3’。
优选的，所述HEPES缓冲溶液的pH为7.0。
本发明检测原理：分别设计两条富含胞嘧啶的DNA链(DNA1和DNA2)，且在这两条链的3’和5’端分别加入两对-GGG-序列。在溶液中Ag+的作用下，形成DNA1-Ag+-DNA2复合物，该复合物中的两条DNA链中的两对-GGG-序列因双链的形成而相互靠近，并进一步形成G四联体DNA模拟酶(辣根过氧化酶类似酶)。该DNA模拟酶能将无色的2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)催化呈深绿色的ABTS˙+，并盖过溶液中的罗丹明B的粉色使溶液颜色为深绿色。而当溶液中有H2S存在时，Ag+和H2S可形成稳定的络合物(Ag2S)，促使DNA1-Ag+-DNA2复合物解体，导致DNA模拟酶的结构被破坏，进而难以完成催化反应，暴露溶液中的罗丹明B的颜色(粉色)，通过溶液从绿色到粉色的转变实现对H2S的裸眼可视化检测。
本发明提供了一种裸眼可视化检测硫化氢的方法。具备以下有益效果：
利用本方法，可对小剂量样本进行H2S含量检测，检测试剂安全稳定易得，检测结果快速、灵敏、裸眼可视，并排除因主观因素导致的观察差异，保证检测结果的准确性。可用于工业、环境、医疗、生活等领域中H2S的检测。
附图说明
图1为本发明的方法原理示意图；
图2为本发明实施例中H2S的检测可行性检测结果示意图；
图3为本发明实施例中不同pH值对H2S检测信号的影响示意图；
图4为本发明实施例中缓冲溶液中不同浓度的H2S的检测结果示意图；
图5为本发明实施例中在10％血清(FBS)中检测不同浓度的H2S示意图。
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。
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。
实施例：如图1所示，本发明实施例提供一种裸眼可视化检测硫化氢的方法，包括以下步骤：
步骤一、将含有10-40mM HEPES缓冲溶液、200-400mM NaCl、50-100mM KCl、0.5-2μM AgNO3、10-100mM罗丹明B、1μM DNA1、1μM DNA2混合，用灭菌后的UP水定容至32μL；
步骤二、向上述溶液中加入0-500μM H2S 5μL，反应15-30min；
步骤三、反应后加入Triton-X-100和Hemin共3μL，反应15-30min；
步骤四、反应后加入10μL含有10-40mM HEPES缓冲溶液、2mM H2O2、1.5mM ABTS的溶液至总体积为50μL，并利用酶标仪记录1-30min时溶液在418nm吸收值，用手机拍照记录溶液颜色变化情况并进行RGB转化。
DNA1的序列为：5’-AAA AAA ACT CTC TCT CTC TCT CTC TCT CAT TAT TTT AGGGCG GG-3’。
DNA2的序列为：5’-GGG CGG GAA AAA TAA TCA CAC ACA CAC ACA CAC ACA CTTTTT TT-3’。
本发明实施例中，胞嘧啶-胞嘧啶(C-C)碱基对可以选择性地捕获银离子(Ag+)，在DNA双链中形成金属介导的碱基对C-Ag+-C，基于以上发现，本发明人分别设计两条富含胞嘧啶的DNA链(DNA1和DNA2)，且在这两条链的3’和5’端分别加入两对-GGG-序列。在溶液中Ag+的作用下，形成DNA1-Ag+-DNA2复合物。该复合物中的两条DNA链中的两对-GGG-序列因双链的形成而相互靠近，并进一步形成G四联体DNA模拟酶(辣根过氧化酶类似酶)。该DNA模拟酶能将无色的2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)催化呈深绿色的ABTS˙+，并盖过溶液中的罗丹明B的粉色使溶液颜色变为深绿色。而当溶液中有H2S存在时，Ag+和H2S可形成稳定的络合物(Ag2S)，促使DNA1-Ag+-DNA2复合物解体，导致DNA模拟酶的结构被破坏，进而难以完成催化反应，最终暴露溶液中的罗丹明B的颜色(粉色)，通过溶液从绿色到粉色的转变实现对H2S的裸眼可视化检测。本发明实施例使用Na2S作为硫化氢供体。
为提高可视化准确度，本发明通过计算机分离实时拍摄照片的RGB通道，将图片转化为RGB图片(R/G→RGB转化)，本方法具有快速、便捷、安全、稳定、裸眼可视的特点，有望实现硫化氢的现场快速检测，为工业、环境、医疗、生活中硫化氢检测带来便捷。
下面结合具体实例对本发明实施例进一步解释：
实例1:H2S的检测可行性
步骤1)将含有10-40mM HEPES缓冲溶液(pH 7.0)、200-400mM NaCl、50-100mMKCl、0.5-2μM AgNO3、10-100mM罗丹明B、1μM DNA1、1μMDNA2混合，用灭菌后的UP水定容至32μL。
步骤2)向上述溶液中加入含有不同浓度的H2S(0及200μM)5μL，反应15-30min。
步骤3)反应后加入Triton-X-100和Hemin共3μL，反应15-30min。
步骤4)反应后加入10μL含有10-40mM HEPES缓冲溶液(pH 7.0)、2mM H2O2、1.5mMABTS的溶液至总体积为50μL，并利用酶标仪记录1-30min时溶液在418nm的吸收值，用手机拍照记录溶液颜色变化情况并进行RGB转化。
结果如图2所示，图2为H2S的检测可行性检测结果示意，其中H2S(-)为不加H2S，H2S(+)为加入200μM的H2S，其中插入的照片为15min时两个样品溶液的颜色。
实例2：反应条件优化
优化HEPES缓冲溶液的反应pH：采用实例1中的检测步骤，其中HEPES缓冲溶液的pH分别为：6.8、7.0、7.4、7.8、8.0。
记录15min时418nm处的吸收值，并进行拟合。
结果如图3所示，图3为不同pH值对H2S检测信号的影响。
实例3:H2S浓度依赖曲线的建立
采用实例1中的检测步骤,其中H2S待测溶液浓度分别为0、0.5、1、5、10、20、40、60、80、100、200、300μM。
记录15min时418nm的吸收值，进行拟合，并对样品拍照，最后将15min时拍的图片进行R/G→RGB转化。
检测结果如图4所示，图4为缓冲溶液中不同浓度的H2S的检测结果示意，(a)为不同浓度的H2S的信号响应，(b)为0-50μM的H2S的标准曲线，(c)为反应15min实时照片及其RGB转化。
实例4：检测生物样本中H2S的含量(10％FBS)
采用实例3中的检测步骤，在10％血清(FBS)溶液中对H2S进行检测。
结果如图5所示，图5：在10％血清(FBS)中检测不同浓度的H2S，(a)为在10％血清(FBS)中不同浓度的H2S的信号响应，(b)为反应15min实时照片及其RGB转化。
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 (4)
1.一种裸眼可视化检测硫化氢的方法，其特征在于：包括以下步骤：
步骤一、将含有10-40 mM HEPES缓冲溶液、200-400 mM NaCl、50-100 mM KCl、0.5-2 μM AgNO3、10-100 mM罗丹明B、1 μM DNA1、1 μM DNA2混合，用灭菌后的UP水定容至32 μL，DNA1与DNA2为两条含胞嘧啶的DNA链，这两条DNA链的3’和5’端分别加入两对-GGG-序列；
步骤二、向上述溶液中加入0-500 μM H2S 5 μL，反应15-30 min；
步骤三、反应后加入Triton-X-100和Hemin共3μL，反应15-30 min；
步骤四、反应后加入10 μL含有10-40 mM HEPES缓冲溶液、2 mM H2O2和1.5 mM ABTS的溶液至总体积为 50 μL，并利用酶标仪记录1-30min时溶液在418 nm吸收值，用手机拍照记录溶液颜色变化情况并进行RGB转化。
2.根据权利要求1所述的一种裸眼可视化检测硫化氢的方法，其特征在于：所述DNA1的序列为：5’-AAA AAA ACT CTC TCT CTC TCT CTC TCT CAT TAT TTT AGG GCG GG-3’。
3.根据权利要求1所述的一种裸眼可视化检测硫化氢的方法，其特征在于：所述DNA2的序列为：5’-GGG CGG GAA AAA TAA TCA CAC ACA CAC ACA CAC ACA CTT TTT TT-3’。
4.根据权利要求1所述的一种裸眼可视化检测硫化氢的方法，其特征在于：所述HEPES缓冲溶液的pH为7.0。
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| WO2016028768A1 (en) * | 2014-08-21 | 2016-02-25 | The University Of Utah Research Foundation | Chemical probes for hydrogen sulfide |
| CN104946752A (zh) * | 2015-06-04 | 2015-09-30 | 中国科学院海洋研究所 | 一种通过肉眼观察对硫酸盐还原菌进行半定量检测的方法 |
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