Abstract
<jats:p> Monitoring protein kinases in their native environment is essential for understanding cellular signaling. This study introduces triangulenium-based peptide biosensors for the real-time detection of the activity of two key protein kinases involved in the T cell immune response: Akt1 and Src. The substrate biosensors incorporate a long-lifetime fluorophore from the triangulenium dye family, which undergoes photoinduced electron transfer (PET) quenching. Upon kinase-mediated phosphorylation, PET is suppressed, resulting in a significant "turn-on" of both fluorescence intensity and fluorescence lifetime (τ <jats:sub>FL</jats:sub> ). This lifetime extension (up to 15 ns) enables the use of fluorescence lifetime imaging microscopy (FLIM) and time-gated filtering to effectively eliminate cellular autofluorescence. <jats:italic toggle="yes">In vitro</jats:italic> and <jats:italic toggle="yes">in extracto</jats:italic> assays confirmed the biosensors' high affinity and specificity for endogenous kinases. Furthermore, <jats:italic toggle="yes">in cellulo</jats:italic> FLIM experiments demonstrated efficient uptake, biocompatibility, and the ability to quantitatively report endogenous Src and Akt1 activities through robust, lifetime-based responses selectively modulated by kinase activators and inhibitors. Crucially, the Akt1 biosensor was successfully applied to isolated primary T cells, where it tracked dynamic signaling changes following T cell receptor (TCR) stimulation using confocal microscopy and flow cytometry. This integrated framework provides a robust, non-invasive platform for quantitative kinase analysis in living cells, with significant implications for immunological profiling and cancer research. </jats:p>