An independent evaluation of the PD20 lumitester for measuring extracellular ATP

Brian King, Gregory Goodey. University College London, London, UK

The hand-held PD20 lumitester was developed by the Kikkoman Corporation for the catering industry, to determine the presence of bacteria on kitchen surfaces, utensils and prepared food. It is primarily designed to measure levels of microbial ATP (mATP) via a luciferase assay, with a linear relationship occurring between the number of bacterial colony forming units and the light generated by this assay. We have carried out an independent evaluation of its sensitivity and specificity for extracellular ATP (eATP), exploring its use in measuring eATP in biological solutions and also testing for eATP on epithelial cells (human skin).

Tap water, filtered water, sterilized water, sterilized and unsterilized salt solutions were tested for contaminating microbial ATP (mATP). Next, the assay sensitivity and optimal incubation time were determined for eATP. Then ADP, AMP, adenosine, CTP, GTP, ITP, UTP, α,βmeATP, pyro-phosphate and inorganic phosphate were assayed and compared to eATP. Additionally, the level of eATP was measured in sweat on human glabrous skin. Each experiment was repeated a minimum of 6 times, the mean±SD were calculated and data compared by Student’s t-test (unpaired).

Filtered water (using a 0.1 micron mesh) possessed the lowest levels of mATP contamination. The PD20 lumitester reacted with linear sensitivity to eATP (10-15 to 10-5 M) either in filtered water or in salt solutions. The optimal incubation time was 60-300s, whereas the minimum discriminatory threshold was 10-13 M. The lumitester is sensitive only to ATP, AMP and PPi. Hand sweat contained the equivalent of 100 nanomolar eATP.

Filtered water is the least contaminated and therefore best solvent for standard curves of eATP. The discriminatory threshold is low (∼0.1 pM) and reliable measurements can be obtained within 60s. Swab-pens contain pyruvate orthophosphate dikinase (which converts AMP to ATP), and so the assay can overcome the loss of ATP to breakdown. The PD20 lumitester can measure eATP in biological samples (e.g. sweat), but also bacterial contamination in biological solutions or on lab equipment and glassware.

Supported by the UCL St Peter’s Trust.