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

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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.