CN108489921B - 一种免双氧水快速检测半胱氨酸的方法 - Google Patents
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Abstract
本发明公开了一种免双氧水快速检测半胱氨酸的方法,其主要是基于新合成的天冬氨酸‑铈纳米材料(Ce‑Asp)具有类氧化酶的活性,在有机底物3,3’5,5’‑四甲基联苯胺(TMB)存在时会发生氧化反应,TMB就会从无色氧化成蓝色,而在加入半胱氨酸的情况下,会抑制其氧化反应,TMB就不会发生颜色变化,本发明即是通过这种比色方法来进行检测半胱氨酸。本发明提供的方法非常简单、特异性强、稳定性好、检测速度快,可开发相关试剂盒、试纸条,能够满足现场快速、准确的检测要求,可应用于食品安全检测、疾病分析、环境分析等领域。
Description
技术领域
本发明涉及化学分析领域,具体涉及一种氨基酸的快速检测方法。本发明属于检测技术领域,
背景技术
半胱氨酸是人体必需氨基酸的一种,是脂肪族中含巯基的极性α-氨基酸。半胱氨酸是一种氨基酸解毒药,参与肝脏细胞的还原和磷脂代谢过程,保护肝细胞受损,促进肝功能恢复,药理作用旺盛。主要用于放射性药物中毒,重金属中毒,锑中毒等,也可用于肝炎,中毒性肝炎,血清病,并可预防肝坏死。目前,已经报道了多种分析技术来检测半胱氨酸,包括光谱学,电化学方法,高效液相色谱法,电感耦合等离子体质谱法等等(Langmμir theAcsJoμrnal of Sμrfaces&Colloids,29(16),5085-92.),但是其中大部分都是耗时的,而且对选择性有限,通常需要昂贵的仪器,不适合现场条件下的快速分析。除此之外还有一些用催化TMB显色的方法来检测Cys(Analyst,2015,140,5251-5256),但这些方法都需要双氧水存在条件下进行,而双氧水特别不稳定,对检测结果准确度有一定的影响。
发明内容
针对以上存在的问题,我们合成了一种新型的天冬氨酸-铈纳米材料,开发了一种无双氧水,免标、快速检测半胱氨酸的新方法。其操作过程简单,成本低,检测结果具有良好的重现性,确保了检测结果的准确度。
为达到上述目的,本发明创造的技术方案如下:
对目标物实现高精准、高灵敏、简单的快速检测方法是分析化学的一个重要发展方向。本发明提供了一种简便快速检测半胱氨酸的方法。本方法先将Ce-Asp与不同浓度Cys混合,在室温孵育10min,再加入TMB,反应10min,TMB的氧化被抑制,至其颜色变化及紫外吸收值也被抑制,其颜色变化强弱及吸收值的大小与Cys浓度呈线性相关,通过观察其颜色变化强度,并通过紫外分光光度仪测量其吸光度即可实现对Cys的快速检测。
本发明具体包括以下步骤:
(1)首先利用Asp和Ce(NO3)3制备Ce-Asp纳米复合材料,基本步骤如下:将1.0mMNaOH和1.0mM Asp加入到6mL醇中并震荡10分钟,接下来,将1.0mMCe(NO3)3溶解在1.0mL去离子水中,将Ce(NO3)3水溶液加入到NaOH和Asp的混合物中并震荡20min,然后6000rpm转速离心6min收集沉淀,并用无水乙醇洗涤三次即可;
(2)然后在0.6mM步骤(1制备的)Ce-Asp纳米复合材料溶液中,加入0-10μM不同浓度的半胱氨酸Cys溶液,孵育10min后再加入150μM TMB,反应10min,反应用9.2mM硫酸终止,再通过紫外分光光度仪测量在451nm处的吸光度值;
(3)TMB,Ce-Asp与不同浓度Cys反应,用硫酸终止反应后通过紫外分光光度仪测定反应体系的吸收光谱,以451nm处最高吸光值强度为输出检测信号,加入Cys后传感体系的吸收值与Cys浓度间存在线性关系,然后以451nm处TMB的紫外吸收值为纵坐标,以Cys浓度为横坐标进行处理做线性关系,据此得到拟合曲线及线性回归方程:y=-0.1374x+1.56574,R=0.9987,检测限为80nM。
本发明所述的方法是一种快速检测半胱氨酸的新方法。相对于现有技术,本发明创造所述的半胱氨酸Cys的检测方法具有以下优势:之前发展的比色检测方法中多数需要用到双氧水,然而双氧水易分解、性质不稳定性,通常需要现用现配,这样限制了相关方法在现场快速检测中的应用。本发明利用Cys,TMB和Ce-Asp的共同作用,所发展的检测方法具有操作简单、免标记、灵敏度高、简便快速和特异性强并且成本低的优势,此方法中Ce-Asp为首次合成,不但合成方法简单快速,合成材料便宜,催化效率高,更重要的是催化TMB不需要双氧水,在无双氧水的情况下可以在十分钟内催化TMB,反应快速,这样更加增加了实验结果的准确性和可靠性。
附图说明
构成本发明创造的附图用来提供对本发明创造的进一步理解,本发明创造的示意性实施例及其说明用于解释本发明创造,并不构成对本发明创造的不当限定。在附图中:
图1为本发明创造实施例所述的材料Ce-Asp的X射线衍射(XRD)光谱图。
图2为本发明创造实施例所述材料氧化以及酸抑制反应提供相关数据。
图3为本发明创造实例所述的TMB反应机理提供相关数据。
图4为本发明创造实施例所述的Ce-Asp氧化TMB所用不同pH的缓冲溶液的影响提供数据。
图5为本发明创造实施例所述的TMB吸光度随Cys浓度变化的变化提供数据。
图6为本发明创造实施例所述的TMB吸光度强度随Cys浓度变化在波长在451nm处吸光度值的线性拟合曲线及方程。
图7为本发明创造实施例所述的检测Cys特异性干扰实验提供数据。
具体实施方式
为了使本发明上述特征和发明创造中优化条件更加清楚和容易理解,下面将结合附图对本发明的实施方式作进一步详细描述。
以下3,3’5,5’-四甲基联苯胺(TMB),硝酸铈-天冬氨酸纳米材料(Ce-Asp)与Cys三者反应体系是200μL(包括TMB,Ce-Asp和不同浓度的Cys).
实施例1
首先,将1mMol NaOH和1mMol Asp加入到6mL醇中并震荡10分钟。接下来,将1mmolCe(NO 3)3溶解在1mL去离子水中。然后,将Ce(NO3)3水溶液加入到NaOH和Asp的混合物中并震荡10min。反应后,通过离心(6000rpm,6min)收集沉淀,并用无水乙醇洗涤三次即可。同样的方法制备不同比例的Ce(NO3)3和Asp合成天冬氨酸-铈纳米材料。再测定其XRD光谱。(a)Asp;(b)1:5Ce-Asp;(c)1:3Ce-Asp;(d)1:1Ce-Asp;(e)Ce(NO3)3。(如图1所示)
实施例2
TMB氧化成蓝色以及终止氧化反应的紫外吸收光谱,(a)150μM TMB;(b)150μM TMB与0.6mMCe-Asp反应;(c)150μM TMB与0.6mMCe-Asp反应后,加上200μM H2SO4终止反应。(如图2所示)
实施例3
TMB氧化反应原理紫外吸收光谱。(a)150μM TMB;(b)0.6mMCe-Asp;(c)150μM TMB与10μMCys混合;(d)0.6mMCe-Asp与10μMCys混合;(e)150μM TMB与0.6mMCe-Asp混合;(f)150μM TMB,0.6mMCe-Asp与10μMCys混合。以上所有反应都用浓硫酸终止反应后,利用紫外分光光度仪测定紫外吸收光谱,波长范围为300nm-600nm。(如图3所示)
实施例4
测定不同pH值对氧化反应的影响。0.6mMCe-Asp与150μM TMB在pH3,4.5,4,5,5.5,6,6.5,7,7.5,8,9的PB缓冲溶液混合孵育10min,测定TMB波长在451nm的紫外吸收值。以位于451nm波长处最大吸收峰为纵坐标,pH值为横坐标做点线图。(如图4所示)
实施例5
TMB氧化反应随加入不同浓度Cys的变化。0.6mMCe-Asp与0-10μM的Cys混合孵育10min,再加入150μM TMB反应10min,反映加入浓硫酸终止,测定波长范围为300nm-600nm紫外吸收光谱。(如图5所示)
实施例6
0.6mMCe-Asp与150μM TMB与0-10μM的Cys混合反应10min,测定TMB在451nm处的吸光度,以TMB在451nm波长处最大吸收值为纵坐标,以不同浓度的Cys为横坐标作图并对其进行线性拟合获得线性方程如下:
y=-0.1374x+1.56574(R2=0.9973)式(1)
实施例7
测定检测Cys反应的特异性。从左到右依次为空白,半胱氨酸,脯氨酸,色氨酸,组氨酸,丝氨酸,甘氨酸,苏氨酸,谷氨酸,精氨酸,丙氨酸,甲硫氨酸,苯丙氨酸,谷氨酰胺,赖氨酸,酪氨酸等作为本实验检测的干扰物质,并研究该方法的特异性。干扰物质浓度均为100μM,分别与0.6mMCe-Asp孵育10min,再加入150μM TMB混合反应10min后,利用紫外分光光度仪测定紫外吸收值。如图6所示,只有10μMCys可以明显抑制TMB吸收光强度,而其他干扰氨基酸没有明显抑制TMB吸收光强度,表明该方法对Cys检测特异性强、灵敏度高、方法非常简单。(如图7所示)
以上所述仅为本发明创造的较佳实施例,并不用于限制本发明创造,凡在本发明创造的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明创造的保护范围之内。
Claims (1)
1.一种免双氧水快速检测半胱氨酸的方法,其特征在于:将Ce-Asp,TMB,和Cys混合,通过观察颜色变化实现对Cys浓度的检测,包括如下步骤:
(1)首先利用Asp和Ce(NO3)3制备Ce-Asp纳米复合材料,基本步骤如下:将1.0mMNaOH和1.0mM Asp加入到6mL醇中并震荡10分钟,接下来,将1.0mMCe(NO3)3溶解在1.0mL去离子水中,将Ce(NO3)3水溶液加入到NaOH和Asp的混合物中并震荡20min,然后6000rpm转速离心6min收集沉淀,并用无水乙醇洗涤三次即可;
(2)然后在0.6mM步骤(1)制备的Ce-Asp纳米复合材料溶液中,加入0-10μM不同浓度的Cys溶液,孵育10min后再加入150μM TMB,反应10min,反应用9.2mM硫酸终止,再通过紫外分光光度仪测量在451nm处的吸光度值;
(3)TMB,Ce-Asp与不同浓度Cys反应,用硫酸终止反应后通过紫外分光光度仪测定反应体系的吸收光谱,以451nm处最高吸光值强度为输出检测信号,加入Cys后传感体系的吸收值与Cys浓度间存在线性关系,然后以451nm处TMB的紫外吸收值为纵坐标,以Cys浓度为横坐标进行处理做线性关系,据此得到拟合曲线及线性回归方程:y=-0.1374x+1.56574,R=0.9987,检测限为80nM。
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