Adenosine triphosphate (ATP) tests are commonly used for cleanliness verification. However, since ATP is hydrolyzed to adenosine diphosphate (ADP) and adenosine monophosphate (AMP), total adenylate (ATP+ADP+AMP, A3) could be more reliable indicators. In this study, a novel enzymatic method to measure A3 was developed based on luciferin-luciferase assay with the combination of two enzymes, pyruvate kinase and pyruvate phosphate dikinase, which can convert ADP and AMP into ATP, respectively. The A3 method afforded equivalent linear calibration curves between the measurement output (Relative Light Units) and each of the amounts of ATP, ADP and AMP. The assays using conventional ATP tests and the A3 tests demonstrated that ATP in hemolysate was decomposed easily, on the other hand, the amounts of A3 were clearly maintained. Regarding sweat samples, the amount of A3 was 20 times higher than that of ATP. Moreover, the investigation for high touch surface samples showed good correlations between the A3 method and viable bacteria counts, and the amount of A3 was about 8 times higher than that of ATP on average. Therefore, the A3 tests appeared to be more reliable for the assessment of sanitation in healthcare settings than the conventional ATP assay.
