ATP measurement in bio-contamination

Abstract

One method to evaluate cell viability is by measuring mitochondrial activity and ATP cell content. Thus, ATP can either provide information on the presence of microbial contamination or be an indicator of cytotoxicity, since it declines very rapidly when cells undergo necrosis or apoptosis. Methods to measure the presence of ATP have been explored, based on different measurement techniques but ATP bioluminescence is presently the most sensitive rapid method for detection of bacterial contamination. Some efforts to improve the assay have adopted protein-engineering techniques to achieve better pH stability and longer wavelength emission. Fortuitously these mutants also show good spectral overlap with the fluorescence proteins that emit in the red region of the spectrum so that a BRET construct can be designed that shifts the emission further to longer wavelengths. With such a format, a ratiometric measurement based on the luciferase/luciferin: fluorescent protein emission can be correlated with ATP concentration. Typically a range of 10 -7 to 10 -10 M ATP can be measured under optimized conditions. Better sensitivity can be demonstrated by amplifying the ATP in an enzyme-recycling pathway, by using, for example, adenylate kinase and polyphosphate kinase. This can result in 10,000-fold amplification of ATP. To put this into context, the average cultivable microbial population in the samples from ordinary rooms is in the range 70-560 CFU ml -1 or 40-3,400 × 10 -15 mol ml -1 ATP. © 2012 Springer Science+Business Media B.V.