Detection by fluorescence microscopy of N-aminopeptidases in bacteria using an ICT sensor with multiphoton excitation: Usefulness for super-resolution microscopy Valverde Pozo, Javier Paredes Martínez, José Manuel Salto Girón, Carmen Herrero-Foncubierta, Pilar Girón González, María Dolores Miguel Álvarez, Delia Cuerva Carvajal, Juan Manuel Álvarez Pez, José María Salto González, Rafael Talavera Rodríguez, Eva María Alanine amino peptidase Fluorescent sensor Bacteria Two-photon excitation STED microscopy Bioimaging This work was financially supported by CTQ2017-85454-C2-1-P, CTQ2017-86125-P and CTQ2017-85658-R (MICIU/AEI/ERDF). JVP is supported by a FPU fellowship (FPU17/04749). CSG was supported by a Research initiation grant (Plan Propio 2018, University of Granada, Spain). We acknowledge the Universidad de Granada (Spain) microscopy central facilities (CIC-UGR). Bacterial proteases are relevant in pathological processes such as the survival, growth and development of microorganisms. In particular alanine amino peptidase (pepN), which is present in Gram (−) bacteria, is the only peptidase responsible for the ATP-independent degradation of cytosolic proteins in E. coli. These peptidases, including pepN, might be useful targets to fight bacterial infections, which are difficult to treat due to the increase in antibiotic resistance, as well as for diagnosis. In this work, we propose a new methodology for the identification of pepN-expressing bacteria by using a specific substrate, namely, DCM−NH−Ala. Substrate hydrolysis by pepN produces a sharp increase in the fluorescence band with peak at 662 nm when excited by a single photon at 480 nm or by two NIR photons (at approximately 800 nm). The emission kinetics are dependent on the intracellular pepN concentrations, which provide a powerful tool for detecting diverse virulent bacteria in a few minutes and with the inherent advantages of two-photon excitation. We resolved the enzymatic kinetics, obtained the Michaelis-Menten parameters (e.g., KM, vmax, and kcatalytic) and studied the photophysics of the released fluorophore DCM–NH2. In addition, DCM–NH2 meets the requirements for use in super-resolution microscopy. In bacteria with high pepN activity, the probe quickly initiates an enzymatic reaction at specific sites located on the bacterial membrane and some structures inside the bacterial body. 2022-02-21T08:36:31Z 2022-02-21T08:36:31Z 2020-10-15 journal article Javier Valverde-Pozo, Jose M. Paredes, Carmen Salto-Giron, Pilar Herrero-Foncubierta, María D. Giron, Delia Miguel, Juan M. Cuerva, Jose M. Alvarez-Pez, Rafael Salto, Eva M. Talavera, Detection by fluorescence microscopy of N-aminopeptidases in bacteria using an ICT sensor with multiphoton excitation: Usefulness for super-resolution microscopy, Sensors and Actuators B: Chemical, Volume 321, 2020, 128487, ISSN 0925-4005, https://doi.org/10.1016/j.snb.2020.128487. http://hdl.handle.net/10481/72924 10.1016/j.snb.2020.128487 eng http://creativecommons.org/licenses/by-nc-nd/3.0/es/ embargoed access Atribución-NoComercial-SinDerivadas 3.0 España Elsevier