Analytical techniques using ATP bioluminescence, which has a high quantum yield and substrate specificity, are widely used in various assays, such as luciferase reporter assays, in the biological sciences. Although most microplate luminometers can be used to measure ATP luminescence with 96-well or 384-well microplates, their ATP detection limits are typically several tens of amol, which is not sufficient for evaluating cell activities and variability within small samples, such as those containing only a few cells. To analyze cell activities at low ATP concentrations, a more sensitive microplate luminometer is required. Therefore, in this study, we developed an automated highly sensitive microplate luminometer that could perform reagent dispensing and bioluminescence measurement with a 96-well microplate within 10 min. ATP bioluminescence was detected by pressing a photomultiplier tube (PMT) against a microplate surface to seal the measured well with the light-receiving surface of the PMT. This enabled a high light collection efficiency and low luminescence crosstalk, defined as the intensity of stray light from an adjacent well. As a result, the ATP detection limit was 0.97 amol, and the luminescence crosstalk was 4.4 × 10-6 . Both values were one order of magnitude better than that of a typical microplate luminometer. In addition, the same gradient linearity of luminescence intensity against the ATP concentration was confirmed for both high and low ATP concentrations, and the dynamic range of our microplate luminometer was 106 . Overall, our findings demonstrated that our novel microplate luminometer may have wide application in biological sciences research.
Keywords: ATP bioluminescence; attomol-level ATP; microplate luminometer.
© 2020 John Wiley & Sons, Ltd.