Ultrasensitive Luminometric ATP Assay with Luminoskan Ascent® and Flash Type ATP Chemistry

Introduction

The luminometric ATP assay is the most common method for conducting quantitative ATP measurements. The assay is based on the following reaction:

When the ATP concentration is measured, D-luciferin and luci-ferase are added in saturated concentration from the assay reagent, and ATP is derived from the unknown sample. With this setting the amount of produced light is directly proportional to the ATP concentration. The assay is normally calibrated with known ATP concentrations, and the concentration of unknown samples is revealed based on the calibration curve using linear regression. The main benefits of the luminometric ATP assay are a very wide dynamic range and high sensitivity. At present there are two common types of ATP assay kits on the market.
One type is based on stable, glow type light production where the reaction produces a constant light production signal over a long period of up to several hours. This is convenient when all reagents are added manually because the signal level can be measured at any time during this stable light production period. This stable reaction is normally achieved by adding inorganic pyrophosphate (PPi) and L luciferin to the assay reagent. Both compounds are inhibitors for the D-luciferin/luciferase reaction and this inhibition converts the light reaction kinetics to a stable, constant format. An important disadvantage of this glow type reaction is that reaction inhibition reduces the sensitivity of the assay.
An alternative assay type uses the ATP flash reaction where there are no inhibitors in the assay reagent. This natural form of D-luciferin/lucife-rase reaction produces instable flash kinetics where the signal maximum is reached in a few seconds after the reaction start, typically 0 to 1 s, and the signal level decreases at high speed. This assay type has much higher sensitivity than the glow type assay, but performing the assay is only possible with instrumentation that has automatic dispensers for reagent addition.