A novel enzymatic method for determination of homocysteine using electrochemical hydrogen sulfide sensor.
A novel enzymatic method for determination of homocysteine using electrochemical hydrogen sulfide sensor.
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DOI：
10.2741/2351
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发表时间：
2007-05
期刊：
影响因子：
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通讯作者：
Dong Zhao;Tsan-Zon Liu;E. Chan;H. Fein;Xueji Zhang
中科院分区：
文献类型：
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作者：
Dong Zhao;Tsan-Zon Liu;E. Chan;H. Fein;Xueji Zhang
同型半胱氨酸是蛋氨酸代谢过程中产生的一种含硫化合物。它在人体血浆中的吸收被认为是心血管疾病和许多其他疾病的原因。提出了一种采用硫化氢传感器与蛋氨酸α、g-裂解酶联用的电化学方法来选择性定量测量同型半胱氨酸。该方法的原理是测量同型半胱氨酸和蛋氨酸 a, g-裂解酶之间的酶促反应中产生的硫化氢。 Tris 缓冲溶液不同 pH 值和室温下的测量灵敏度在 pH 6.5 时达到峰值 275 pA/mM，检测限为 150 nM（基于 3 秒截止）。在 pH 6.5 下进行线性测量，同型半胱氨酸浓度范围为 0.5 至 200 mM，该范围比人血浆总同型半胱氨酸水平 5 至 100 mM 更宽，实验的回归分析得出 R2=0.9987。该酶还对 pH 7.5 的 tris 缓冲溶液中的同型半胱氨酸表现出最快的响应，电流在 134 秒时达到最大值。对几种常见试剂进行了干扰试验，发现半胱氨酸和蛋氨酸是引入测量问题的主要两种物质。针对这一干扰问题的解决方案，在前期试验的基础上进行了深入的探索和讨论。还进行了针对几种酶浓度的实验灵敏度。
Homocysteine is a sulfur-containing compound produced during metabolism process of methionine. Its uptake in human plasma is believed to be the cause of cardiovascular diseases and many other diseases. An electrochemical method was proposed for selective and quantitative measurement of homocysteine by employing hydrogen sulfide sensor coupled with methionine a, g-lyase. The principle of this method is to measure the evolved hydrogen sulfide from the enzymatic reaction between homocysteine and methionine a, g-lyase. The sensitivities of the measurements at different pH values of the tris buffer solutions and at room temperature peaked to 275 pA/mM at pH 6.5 with detection limit of 150 nM (based on 3 s cutoff). The linearity measurements at pH 6.5 were performed for the homocysteine concentrations range from 0.5 to 200 mM, which is wider than the human blood plasma total homocysteine level of 5 to 100 mM, and the regressive analysis of the experiments gave R2=0.9987. The enzyme also showed the fastest response to homocysteine in the tris buffer solution of pH 7.5 with the current approaching its maximum at 134 seconds. The interference tests against several common agents were carried out, and found that cysteine and methionine were the major two species to introduce measurement problem. The solution to this interference problem was explored and discussed thoroughly based on the preliminary tests. The sensitivities of the experiments against several enzyme concentrations were also performed.