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This study introduces triangulenium\u2010based 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\u2010lifetime fluorophore from the triangulenium dye family, which undergoes quenching by photoinduced electron transfer (PET) from different amino acid side groups. Upon kinase\u2010mediated phosphorylation, the PET quenching is significantly suppressed, increasing both fluorescence intensity and fluorescence lifetime (\u03c4\n                    <jats:sub>FL<\/jats:sub>\n                    ). This lifetime extension (up to 15\u00a0ns) enables the use of fluorescence lifetime imaging microscopy (FLIM) and time\u2010gated 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\u2011based 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\u2010invasive platform for quantitative kinase analysis in living cells, with significant implications for immunological profiling and cancer research.\n                  <\/jats:p>","DOI":"10.1002\/smll.75317","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T08:30:57Z","timestamp":1787214657000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Triangulenum\u2010Based Biosensor Platform for Mapping Kinase Activity in T Cells"],"prefix":"10.1002","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5573-0631","authenticated-orcid":false,"given":"Carmen","family":"Salto\u2010Giron","sequence":"first","affiliation":[{"name":"Nanoscopy\u2010UGR Laboratory. 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Departamento de Fisicoqu\u00edmica Unidad de Excelencia de Qu\u00edmica Aplicada a Biomedicina y Medioambiente Facultad de Farmacia Campus Cartuja Universidad De Granada  Granada Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-9511-9306","authenticated-orcid":false,"given":"Antonio","family":"Reinoso","sequence":"additional","affiliation":[{"name":"Departamento De Bioqu\u00edmica y Biolog\u00eda Molecular II Unidad de Excelencia de Qu\u00edmica Aplicada a Biomedicina y Medioambiente Facultad De Farmacia Campus Cartuja Universidad De Granada  Granada Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5371-8671","authenticated-orcid":false,"given":"Laura","family":"Espinar\u2010Barranco","sequence":"additional","affiliation":[{"name":"Department of Medicine Translational Transplant Research Center Immunology Institute Icahn School of Medicine at Mount Sinai  New York New York USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7044-3611","authenticated-orcid":false,"given":"Rafael","family":"Salto","sequence":"additional","affiliation":[{"name":"Departamento De Bioqu\u00edmica y Biolog\u00eda Molecular II Unidad de Excelencia de Qu\u00edmica Aplicada a Biomedicina y Medioambiente Facultad De Farmacia Campus Cartuja Universidad De Granada  Granada Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9620-6668","authenticated-orcid":false,"given":"Amparo","family":"Navarro","sequence":"additional","affiliation":[{"name":"Departamento De Qu\u00edmica F\u00edsica y Anal\u00edtica Facultad De Ciencias Experimentales Universidad De Ja\u00e9n  Ja\u00e9n Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1804-8136","authenticated-orcid":false,"given":"Miguel","family":"Fribourg","sequence":"additional","affiliation":[{"name":"Department of Medicine Translational Transplant Research Center Immunology Institute Icahn School of Medicine at Mount Sinai  New York New York USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1905-4183","authenticated-orcid":false,"given":"Angel","family":"Orte","sequence":"additional","affiliation":[{"name":"Nanoscopy\u2010UGR Laboratory. 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