CN105891182B - 一种对过氧化氢酶定量的方法 - Google Patents
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- YVWNBNDYTKPZFP-UHFFFAOYSA-N 2-oxo-2-phenoxyacetic acid Chemical compound OC(=O)C(=O)OC1=CC=CC=C1 YVWNBNDYTKPZFP-UHFFFAOYSA-N 0.000 claims abstract description 12
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Abstract
一种对过氧化氢酶定量的方法，属于生物检测技术领域。本发明包括使用以下三种化学物质：过氧化氢、苯基草酸酯和9,10‑二苯基蒽荧光染料；利用过氧化氢和苯基草酸酯之间的化学反应产生的能量激发9,10‑二苯基蒽产生荧光信号，而过氧化氢酶则会抑制这种化学反应，导致荧光信号的减弱或猝灭，将荧光信号转换为电信号，通过电信号的强度测定过氧化氢酶的含量。本发明提供一种可以快速准确的对过氧化氢酶定量的方法，灵敏度高、操作方便，检测范围宽，能够对痕量及高浓度过氧化氢酶进行精确定量。
Description
技术领域
本发明涉及一种快速测定过氧化氢酶含量的方法，属于生物检测技术领域。
背景技术
生物体在新陈代谢过程中,会产生一些有害物质,这些有害物质只有被快速分解清除,生物体才能维持正常的生命活动。过氧化氢是生物体产生的一种有害物质,氧化性极强，对生物体的危害很大，需要被快速的分解。过氧化氢酶能催化过氧化氢分解,并产生氧气，与生物的呼吸作用、光合作用及生长素的氧化等都有密切关系。在生物体生长活动过程中，过氧化氢酶的活性不断发生变化，过氧化氢酶的浓度，可以反映某一时期生物体内代谢的变化。例如，在对污水进行生物处理过程中，过氧化氢酶活性与活性污泥中的微生物呼吸强度和活动状况相关，在一定程度上可反映污水处理中微生物处理污水的效能，直接影响出水水质。土壤中的过氧化氢酶含量也直接反应了土壤的呼吸强度和土壤中微生物的活动强度。
常用的过氧化氢酶的测定方法包括测压法和滴定法。测压法是测定过氧化氢分解时析出的氧量，方法简单，但易受环境中含氧的影响，准确性较差。滴定法是定量滴定酶促反应后剩余的过氧化氢量，准确度相对较高，但操作较复杂。
如何快速准确的对食品、工业、环境及生命体系中的过氧化氢酶进行定量，促成了本发明的形成。本发明通过寻求荧光信号变化强度与过氧化氢酶含量间的线性关系范围，间接定量检测环境中的过氧化氢酶的含量。通过大量的实验数据，确定本发明的检测目标的范围，同时保证检测的精确度。
分类号G01N21/33(2006.01)I公开了一种活性污泥过氧化氢酶活性的测定方法，该发明“为了通过测定活性污泥过氧化氢酶活性，揭示过氧化氢酶与活性污泥之间的内在关系，定量分析和评价活性污泥微生物的氧化还原酶状况，从分子水平表征活性污泥的内在运行特征，采用紫外分光光度法，利用过氧化氢在245nm处有强烈吸收的特性，建立了活性污泥过氧化氢酶活性测定方法。本发明的积极效果是简单易行，安全可靠，成本较低，测定精度高，重现性好，可实现活性污泥中过氧化氢酶活性的快速测定。”
分类号G01N21/64(2006.01)I公开了一种基于金纳米团簇探针的过氧化氢酶荧光测定方法，其涉及“以N-乙酰-L-半胱氨酸保护的金纳米团簇为荧光探针的过氧化氢酶测定方法，其特征是利用Fe2+催化H2O2产生羟自由基使金纳米团簇的荧光发生猝灭，而过氧化氢酶可催化H2O2分解生成H2O和O2，抑制金纳米团簇荧光的猝灭，从而表现出荧光发射光谱特征的变化，可以用于过氧化氢酶的检测。在0.01～0.3U/mL范围内荧光强度变化值ΔF650与过氧化氢酶浓度呈线性关系，检测限为0.002U/mL。本发明灵敏度高，重现性好，可用于食品、工业、环境及生命体系中过氧化氢酶的测定。”
本发明本提供一种快速准确测定过氧化氢酶的方法，利用过氧化氢酶与过氧化氢的酶促反应，间接定量过氧化氢酶的含量。
发明内容
为了克服常规检测准确性较差，操作较复杂的问题，发明一种快速准确测定过氧化氢酶的方法。本发明通过寻求荧光信号变化强度与过氧化氢酶含量间的线性关系范围，间接定量检测环境中的过氧化氢酶的含量。通过大量的实验数据，确定本发明的检测目标的范围，同时保证检测的精确度。
一种对过氧化氢酶定量的方法，其特征在于，包括使用以下三种化学物质：过氧化氢、苯基草酸酯和9,10-二苯基蒽荧光染料；首先，利用过氧化氢和苯基草酸酯反应，过氧化氢对苯基草酸酯的羰基亲核进攻，生成能产生高能量的双氧基环状中间体二氧杂环丁二酮；中间体分解将能量传递给受体荧光分子，使之处于激发状态；激发态的9,10-二苯基蒽不稳定，分解回到稳定基态的同时，发射荧光，而过氧化氢酶能催化H2O2分解生成H2O和O2，可导致荧光信号的减弱或猝灭；
在0.01～5U/mL范围内，荧光信号变化强度与过氧化氢酶的含量呈负相关的线性关系，表现出荧光发射光谱特征的变化；
将发出的荧光信号转换为电信号，通过传感器直接指示过氧化氢酶的含量；信号越强，过氧化氢酶的含量越低，反之，含量越高；
过氧化氢酶在0.01～5U/mL浓度范围内，对应的电信号强度为47.73～52.56 mv/m，过氧化氢酶浓度为零时，对应的电信号强度为60mv/m；检测限为0.001U/mL；
具体步骤如下：
（1）取待检样品50mL，调pH至8～8.5，向样品中加入2mL重铬酸钾，摇匀；
（2）向上述混合液中加入0.3%的过氧化氢溶液5毫升；
（3）再加入浓度为0.05mol/L的苯基草酸酯溶液0.5毫升和9,10-二苯基蒽溶液1.0毫升，摇匀30s，激发荧光；
（4）将反应混合液置于荧光转换器，再通过信号转换器将荧光信号转化成电信号，根据输出的电信号强度定量样品中过氧化氢酶的浓度。
本发明可以快速准确的对过氧化氢酶定量，灵敏度高、操作方便；检测范围宽，能够对痕量及高浓度过氧化氢酶进行精确定量。
具体实施方式
本发明一种对过氧化氢酶定量的方法，其特征在于，包括以下三种化学物质：过氧化氢、苯基草酸酯和9,10-二苯基蒽荧光染料；首先，利用过氧化氢和苯基草酸酯反应，过氧化氢对苯基草酸酯的羰基亲核进攻，生成能产生高能量的双氧基环状中间体二氧杂环丁二酮；中间体分解将能量传递给受体荧光分子，使之处于激发状态；激发态的9,10-二苯基蒽不稳定，分解回到稳定基态的同时，发射荧光，而过氧化氢酶能催化H2O2分解生成H2O和O2，可导致荧光信号的减弱或猝灭；
在0.2-3U/mL范围内，荧光信号变化强度与过氧化氢酶的含量呈负相关的线性关系，表现出荧光发射光谱特征的变化；
将发出的荧光信号转换为电信号，通过传感器直接指示过氧化氢酶的含量；信号越强，过氧化氢酶的含量越低，反之，含量越高；
过氧化氢酶在0.2-3U/mL浓度范围内，对应的电信号强度为49.66～52.37 mv/m，过氧化氢酶浓度为零时，对应的电信号强度为60mv/m；检测限为0.001U/mL；
具体步骤如下：
（1）取待检样品50mL，调pH至8，向样品中加入2mL重铬酸钾，摇匀；
（2）向上述混合液中加入0.3%的过氧化氢溶液5毫升；
（3）再加入浓度为0.05mol/L的苯基草酸酯溶液0.5毫升和9,10-二苯基蒽溶液1.0毫升，摇匀30s，激发荧光；
（4）将反应混合液置于荧光转换器，再通过信号转换器将荧光信号转化成电信号，根据输出的电信号强度定量样品中过氧化氢酶的浓度。
Claims (1)
1.一种对过氧化氢酶定量的方法，其特征在于，包括使用以下三种化学物质：过氧化氢、苯基草酸酯和9,10-二苯基蒽荧光染料；首先，利用过氧化氢和苯基草酸酯反应，过氧化氢对苯基草酸酯的羰基亲核进攻，生成能产生高能量的双氧基环状中间体二氧杂环丁二酮；中间体分解将能量传递给受体荧光分子，使之处于激发状态；激发态的9,10-二苯基蒽不稳定，分解回到稳定基态的同时，发射荧光，而过氧化氢酶能催化H2O2分解生成H2O和O2，可导致荧光信号的减弱或猝灭；
过氧化氢酶在0.01～5U/mL范围内，荧光信号变化强度与过氧化氢酶的含量呈负相关的线性关系，表现出荧光发射光谱特征的变化；
将发出的荧光信号转换为电信号，通过传感器直接指示过氧化氢酶的含量；信号越强，过氧化氢酶的含量越低，反之，含量越高；
过氧化氢酶在0.01～5U/mL浓度范围内，对应的电信号强度为47.73～52.56 mv/m，过氧化氢酶浓度为零时，对应的电信号强度为60mv/m；检测限为0.001U/mL；
具体步骤如下：
（1）取待检样品50mL，调pH至8～8.5，向样品中加入2mL重铬酸钾，摇匀；
（2）继续加入0.3%的过氧化氢溶液5毫升；
（3）再加入浓度为0.05mol/L的苯基草酸酯溶液0.5毫升和9,10-二苯基蒽溶液1.0毫升，摇匀30s，激发荧光；
（4）将反应混合液置于荧光转换器，再通过信号转换器将荧光信号转化成电信号，根据输出的电信号强度定量样品中过氧化氢酶的浓度。
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| CN101743467A (zh) * | 2007-06-04 | 2010-06-16 | 马里兰大学生物技术研究所 | 微波腔中的荧光显微镜 |
| CN102861334A (zh) * | 2012-10-11 | 2013-01-09 | 南京大学 | 肿瘤信号响应的主动治疗纳米光动力药物载体及制法 |
| CN103645152A (zh) * | 2013-12-12 | 2014-03-19 | 尹军 | 一种活性污泥过氧化氢酶活性的测定方法 |
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