Fluorescence Lifetime FLT

While open-chain cyanines such as Cy5, Cy5.5, or Alexa 647 typically exhibit small fluorescence lifetime changes upon binding to biomolecules, squaraine dyes, in general, demonstrate pronounced lifetime changes upon binding to large-molecular-weight analytes [17, 18], which can be used for FLT-based assays. 

The same publication indicated that fluorescence lifetimes of compounds from the compound collection of Pfizer classified as problematic due to their autofluorescence characteristics resulted in false positive results in many FI-based assays. For most compounds, the fluorescence lifetimes were below 1 ns. Thus, FLT measurements with a reporter fluorophore displaying a lifetime significantly longer than 1 ns are suitable for application in protease assays for compound testing. However, for applications under initial velocity conditions with a substrate turnover below 20%, fluorophores with lifetimes of a few nanoseconds are still critical because the dynamic range of the assay is then too low with lifetimes below 1 ns. 

Protease assays based on the FLT of PT14 are especially attractive due to the long lifetime of the dye of 14 ns that allows the discrimination of the lifetimes of short-lived fluorescent compounds from that of PT14. Thus, the rate of false-positive and -negative results can be reduced. The major... 

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