Ratiometric Two-Photon Near-Infrared Probe to Detect DPP IV in Human Plasma, Living Cells, Human Tissues, and Whole Organisms Using Zebrafish Valverde Pozo, Javier Paredes Martínez, José Manuel Widmann, Thomas J. Griñán Lisón, Carmen García Rubiño, María Eugenia Marchal Corrales, Juan Antonio Álvarez Pez, José María Talavera Rodríguez, Eva María DPP IV Ratiometric fluorescent sensor NIR probe Two-photon excitation Bioimaging DPP IV, otherwise known as CD26 lymphocyte T surface antigen, is a transmembrane glycoprotein also found in circulation in the blood. It plays an important role in several processes like glucose metabolism and T-cell stimulation. Moreover, it is overexpressed in renal, colon, prostate, and thyroid human carcinoma tissues. It can also serve as a diagnostic in patients with lysosomal storage diseases. The biological and clinical importance of having readouts for the activity of this enzyme, in physiological and disease conditions, has led us to design a near-infrared (NIR) fluorimetric probe that also has the characteristics of being ratiometric and excitable by two simultaneous NIR photons. The probe consists of assembling an enzyme recognition group (Gly-Pro) (Mentlein, 1999; Klemann et al., 2016) on the two-photon (TP) fluorophore (derivative of dicyanomethylene- 4H-pyran, DCM-NH2) disturbing its NIR characteristic internal charge transfer (ICT) emission spectrum. When the dipeptide group is released by the DPP IV-specific enzymatic action, the donor−acceptor DCM-NH2 is restored, forming a system that shows high ratiometric fluorescence output. With this new probe, we have been able to detect, quickly and efficiently, the enzymatic activity of DPP IV in living cells, human tissues, and whole organisms, using zebrafish. In addition, due to the possibility of being excited by two photons, we can avoid the autofluorescence and subsequent photobleaching that the raw plasma has when it is excited by visible light, achieving detection of the activity of DPP IV in that medium without interference. 2023-03-24T08:37:03Z 2023-03-24T08:37:03Z 2023-02-27 info:eu-repo/semantics/article ACS Sens. 2023, 8, 3, 1064–1075. [https://doi.org/10.1021/acssensors.2c02025] https://hdl.handle.net/10481/80796 10.1021/acssensors.2c02025 eng http://creativecommons.org/licenses/by/4.0/ info:eu-repo/semantics/openAccess Atribución 4.0 Internacional American Chemical Society