A Triangulenum‐Based Biosensor Platform for Mapping Kinase Activity in T Cells

ABSTRACT Monitoring protein kinases in their native environment is essential for understanding cellular signaling. This study introduces triangulenium‐based peptide biosensors for detection of the activity of two key protein kinases involved in T cell immune response: Akt1 and Src. The substrate biosensors incorporate a long‐lifetime fluorophore from the triangulenium dye family, which undergoes quenching by photoinduced electron transfer (PET) from different amino acid side groups. Upon kinase‐mediated phosphorylation, the PET quenching is significantly suppressed, increasing both fluorescence intensity and fluorescence lifetime (τ FL ). 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. In vitro and in extracto assays confirmed the biosensors' high affinity and specificity for endogenous kinases. FLIM experiments in HeLa cells demonstrated efficient uptake, biocompatibility, and quantitative reporting of endogenous Src and Akt1 activities through robust, lifetime‑based responses selectively modulated by kinase activators and inhibitors. Crucially, the Akt1 biosensor tracked dynamic signaling changes in isolated primary T cells following T cell receptor 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.