Dr Wayne Bowen, CSO, TTP Labtech
The road well-travelled
The introduction of fluorescence-based analytical methods revolutionised the landscape of visualising biological systems in a flurry of new reagents and beautiful images. As we moved away from radiochemical labelling to fluorescence-based systems, researchers and companies benefited from an astonishing increase in specificity, sensitivity and speed. This was especially apparent in high throughput screening (HTS) and its application in the search for therapeutic targets. Until recently assay interferences such a quenching, auto-fluorescence and light scattering have been major concerns, affecting data validity through the generation of false positive results. With HTS always pushing for faster, more accurate results over an ever larger number of samples, the introduction of errors along the way can rapidly condemn and entire dataset resulting in wasted time and money.
Fluorescence lifetime (FLT) can be viewed as an alternative reporter system since it largely mitigates such issues. In brief, FLT is the period of time between excitation of a fluorophore and its return to a ground state energy level. This lifetime of just a few nanoseconds is highly dependent upon the local microenvironment, for example the pH or the presence of other ions, and thus offers a unique insight into biochemical systems. People have been making use of FLT assays for HTS for well over a decade now, yet the technique has never really gained widespread acceptance.
Can FLT deliver for HTS?
The obvious potential of FLT assays has not been overlooked; instead, companies such as TTP Labtech have been developing robust, cost-effective technologies in the form of HTS-compatible readers. Together with forming alliances with companies that produce high quality reagents, TTP Labtech can fully exploit this potential. The ameon® microplate reader that has been developed here at TTP Labtech represents the next generation of FLT reader providing a combination of speed, precision and data quality that can readily be exploited in HTS workflows. The system is unique in offering real-time decay curve analysis (RT-DCA) which employs a proprietary digitiser that is capable of recording the entire fluorescence decay curve in nanoseconds for each laser pulse. This allows for rapid read times of approximately two minutes for a 1536-well microplate due the higher levels of photons that the system is able to make use of. This improves both statistical confidence and the speed of the HTS process.
The development of new dyes with remarkably long fluorescence lifetimes is similarly making FLT assays in drug discovery more attractive. PT14, PT22 (GE Healthcare) and 9AA (Almac Group, FLEXYTETM) have lifetimes between 14 and 22 ns. Due to these lifetimes greatly exceeding those of the compounds used in pharmaceutical companies’ screening decks, an automated correction of assay data by utilisation of the differences in FLTs between dyes and compounds is possible.
FLT technology is now more efficient, cost-effective and is even relatively easier than similar time-resolved technologies (HTRF and Alphascreen) for protein kinase assays. Technological advancements have pushed FLT assays to the front of HTS and is now poised to take its place amongst the most valued.