A Highly Sensitive Biosensor for ATP Using a Chimeric Firefly Luciferase

Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications based on the detection of the enzymes or using the proteins to measure ATP levels. Based on studies of chimeric luciferases, we engineered a novel luciferase called PLG2 that has enhanced specific activity, and thermal and pH stability compared to the commonly used Photinus pyralis luciferase. We present here protocols for preparing a single assay mixture containing PLG2 that can be used to readily detect femtomole levels of ATP. Our methodology can be used with a variety of samples, including human and bacterial cells, where measurements of ATP can be used as a biosensor for the detection of viable cells.