Masahiro Okanojo

Attomol-level ATP bioluminometer for detecting single bacterium: ATP calibration curve is linear from 0 to 5 amol

We have developed an automated high-sensitive ATP bioluminometer for detecting single bacterium. The apparatus consists of a tube rack for setting reagents and samples, two washing baths for preventing sample carry-over from dispenser nozzle, and x-, y-, z- actuators for moving the dispenser, and an high-sensitive optical system. The reaction tube was selected to reduce the background signal intensities for the ATP bioluminescence measurement. The background signal intensity of the reaction tube was 18 RLU, which is almost the same as the dark counts of the photomultiplier (16 RLU). The ATP calibration curve was linear from 0 to 5 amol (its slope = 22.4 RLU/amol and 3.3 SD of the blank sample signal = 17.9 RLU), and the detection limit of 0.8 amol was obtained. The relationship between intracellular ATP and CFU in Escherichia coli (ATCC25922) was kept linearity from 0 to 20 CFU, and the intracellular ATP (amol) per CFU was calculated to be 3.3 amol/CFU (R2 = 0.9713). Moreover, the relationship between intracellular ATP and CFU in Staphylococcus aureus (ATCC25923) was also kept linearity from 0 to 30 CFU, and the amol/CFU was calculated to be 1.6 amol/CFU (R2 = 0.9847). The automated ATP bioluminometer has ultra-high sensitivity and will be a powerful tool for measuring ATP luminescence derived from small number of bacteria.