Kuhn, S. M.; Nerli, E.; Liu, J.; Kaufmann, S.; Barahtjan, P.; Buitrango-Arango, M.; Geertsma, E. R.; Mateus, R.; Grapin-Botton, A.; Nadler, A.

Genetically encoded fluorescent biosensors are widely used to monitor small molecule and ion levels in living cells. Quantitative FRET and FLIM sensors and highly sensitive intensiometric sensors have been developed for many analytes. Notwithstanding notable advances over the last years, a universal high-performance sensor design for absolute quantification that does not require specialized equipment has yet to be developed. We here report the GEQO platform of quantitative biosensors featuring calcium, ATP, cAMP, and organelle-specific variants. We used GEQO sensors to follow calcium and cAMP transients in immortalised cells, human pancreatic progenitor cells, and zebrafish embryos. We show that GEQO-based absolute quantification allows to account for analyte buffering and retains information in time trace data lost during relative quantification. GEQO biosensors will enable quantitative analyte measurements across a wide range of imaging platforms, a key prerequisite for diagnostic applications and quantitative approaches in basic cell biology.