Development of a highly sensitive microplate luminometer using ATP bioluminescence.

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.

A Rapid ATP Bioluminescence-based Test for Detecting Levofloxacin Resistance Starting from Positive Blood Culture Bottles.

Administering appropriate antimicrobial therapy as early as possible is important for rescuing bacteremic patients. Therefore, rapid antimicrobial susceptibility tests in positive blood culture specimens have been diligently sought. Adenosine triphosphate (ATP) bioluminescence-based methods have been used for rapid antimicrobial susceptibility tests. However, blood culture specimens have not been examined in many studies, possibly due to abundant intracellular ATP in blood corpuscles resulting in false-susceptible results. In this study, we developed a rapid ATP bioluminescence-based method for detecting antibiotic resistance starting from positive blood culture. To minimize background ATP originating from blood corpuscles, specimens were centrifuged and the supernatant diluted with broth, and an ATP-eliminating reagent was then added to the bacterial suspension at the beginning of incubation. This newly devised procedure reduced the background ATP by more than five orders of magnitude. In a pilot study using levofloxacin, no false-susceptible results were observed in 15 clinical specimens. Furthermore, the results indicated that the rapid method provided additional information about bacterial activities with high resolution, in contrast to the less-thorough findings with the conventional turbidity method. Therefore, our approach will contribute to the treatment of infectious diseases as a rapid antimicrobial susceptibility test.