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

ATP在代谢的各个方面都起着核心作用，因此ATP检测的任何分析在纯生物化学和应用生物化学中都非常重要。使用萤火虫荧光素酶的ATP的生物发光测定是一种成熟的技术。这种assa在商业上被用作快速卫生监测，最近被用作对抗生物恐怖主义的技术。然而，该测定技术具有检测极限（例如每次测定约10 μ<th>菌落形成单位[CFU]的大肠杆菌），这对于一些商业应用不够灵敏。我们设计了一种超灵敏的ATP生物发光检测方法，采用腺苷酸激酶（ADK）作为第一个将AMP + ATP转化为两分子ADP的酶，多磷酸（聚）激酶（PPK）作为第二个将ADP转化为ATP的酶。在该反应中，过量的AMP和息肉分别用于驱动ADK和PPX平衡朝向ADP和AT形成。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，与从存在的初始ATP获得的生物发光信号相比，产生大的生物发光信号。这种测定技术使我们能够检测到饮用水中每毫升一个CFU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.