Comparison of Fluorescence Lifetime and Fluorescence Intensity Readouts using a Homogeneous Protease-assay
Poster Oct 14, 2005
Joachim Janda, Michael Busch, Christoph Wenger and Ralf Thiericke
In this work two measurement parameters are compared, the fluorescence intensity (FI) and fluorescence lifetime (FLT), which could be acquired efficiently in a multi parameter readout from one and the same measurement.
The biochemical reaction being investigated consists of a fluorescent labelled enzyme-substrate (case in as a biopolymer carrying BODIPY FL), which is cleaved by the protease a-chymotrypsin.
In the beginning of the assay the fluorescence signal is internally quenched as a result of energy transfer interactions among the BODIPY labels. The continuous fragmentation of the biopolymer backbone in consequence of enzymatic degradation leads to a rise of the fluorescence signal while the quenching effects decrease. In the assay this is expressed in an increase of both, intensity and lifetime.
When there is a need to quickly analyze samples using a number of different PCR assays, it is likely that optimal conditions for each assay will not be the same. First, different assays often will require different annealing temperatures for their primers. In addition, amplicons may be designed to be of different lengths and therefore require varying durations of the extension step.READ MORE