A multienzyme bioluminescent time-resolved pyrophosphate assay
A multienzyme bioluminescent time-resolved pyrophosphate assay
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DOI：
10.1016/j.ab.2007.04.023
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发表时间：
2007-08-15
影响因子：
2.9
通讯作者：
Golovlev, Val
中科院分区：
文献类型：
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作者：
Sun, Ye;Jacobson, K. Bruce;Golovlev, Val
我们开发了一种高灵敏度的测定方法，用于测定5'-三磷酸腺苷（ATP）污染样品中的无机焦磷酸盐（PPi）。该分析基于发光动力学的时间分辨测量，并实现多种酶将PPi转化为ATP， ATP反过来用于产生光并水解PPi以测量稳态背景发光。提出了一种描述发光动力学和优化检测混合物组成的理论模型。我们发现该模型与实验结果非常吻合。我们已经开发并评估了从atp污染样品中获得的发光动力学中测量PPi的两种算法。第一种算法被认为是分析长时间（即3-5分钟）动力学的首选方法。在实验过程中控制酶的活性；在atp污染的样品中，PPi检测的灵敏度约为7 pg/ml或15 pM PPi。第二种算法用于分析短的，即小于1分钟的发光动力学。它具有约20 pM PPi检测灵敏度，可能是微孔板格式检测的更好选择，其中需要较短的测量时间。PPi测定主要用于RNA表达分析，但它也可用于需要在atp污染样品中进行高灵敏度PPi检测的各种应用。(C) 2007爱思唯尔公司版权所有。
We have developed a high-sensitivity assay for measurement of inorganic pyrophosphate (PPi) in adenosine 5'-triphosphate (ATP)contaminated samples. The assay is based on time-resolved measurements of the luminescence kinetics and implements multiple enzymes to convert PPi to ATP that is, in turn, utilized to produce light and to hydrolyze PPi for measurement of the steady state background luminescence. A theoretical model for describing luminescence kinetics and optimizing composition of the assay detection mixture is presented. We found that the model is in excellent agreement with the experimental results. We have developed and evaluated two algorithms for PPi measurement from luminescence kinetics acquired from ATP-contaminated samples. The first algorithm is considered to be the method of choice for analysis of long, i.e., 3-5 min, kinetics. The activity of enzymes is controlled during the experiment; the sensitivity of PPi detection is about 7 pg/ml or 15 pM of PPi in ATP-contaminated samples. The second algorithm is designed for analysis of short, i.e., less than 1-min, luminescence kinetics. It has about 20 pM PPi detection sensitivity and may be the better choice for assays in microplate format, where a short measurement time is required. The PPi assay is primarily developed for RNA expression analysis, but it also can be used in various applications that require high-sensitivity PPi detection in ATP-contaminated samples. (C) 2007 Elsevier Inc. All rights reserved.