Development of an ultra-sensitive ATP assay by an enzymatic ATP amplification reaction.

三磷酸腺苷在代谢的各个方面都起着核心作用，因此检测三磷酸腺苷的方法在生物化学和应用生物化学中都具有重要意义。利用萤火虫荧光素酶测定三磷酸腺苷的生物发光是一种成熟的技术。这种ASSA在商业上被用作一种快速的Hyginiene监测，最近还被用作一种打击生物恐怖主义的技术。然而，这种检测技术有一个检测极限(例如每次检测大约10个菌落形成单位[CFU])，这对于一些商业应用来说不够灵敏。在这里，我们设计了一种超灵敏的生物发光分析方法，以腺苷酸激酶(ADK)为第一酶，将AMP+ATP转化为两个ADP分子，并以多聚磷酸(Polyp)激酶(PPK)为第二酶，将ADP转化为ATP。在该反应中，过量的AMP和POLP分别被用来驱动ADK和PPX向ADP和AT的形成平衡。如果存在一个ATP分子，则第一个ADK反应开始，内源性AMP转化为ATP，产生比最初存在的ATP更大的生物发光信号。这项检测技术使我们能够检测到饮用水中每毫升一个菌落单位的细菌污染。

ATP plays a central role in all aspects of metabolism and so any assays for ATP detection are of great importance in both pur and applied biochemistry. The bioluminescence assay of ATP using firefly luciferase is a well established technique. This assa is commercially used as a rapid hyginiene monitoring and recently as a technology against bioterrorism. However, this assay technique has a detection limit (for instance approximately 10^<th> colony-forming unit [CFU] of Escherichia coil per assay), which is not sensitive enough for some commercial applications. Here we designed an ultra-sensitive bioluminescence assay for ATP by employing adenylat kinase (ADK) as the first enzyme to convert AMP + ATP to two molecules of ADP and polyphosphate (polyp) kinase (PPK) as the second enzyme to convert ADP to ATP. In this reaction, excess AMP and polyp are used to drive ADK and PPX equilibrium toward ADP and AT formation, respectively. If one molecule of ATP present, the first ADK reaction starts and endogenous AMP is converted to ATP, producing a large bioluminescence signal compared to that obtained from the initial ATP present. This assay technique enabled us to detect bacterial contamination of one CFU per ml in a drinking water.