A highly photostable and versatile two-photon fluorescent probe for the detection of a wide range of intracellular nitric oxide concentrations in macrophages and endothelial cells

Gold nanoparticle-based two-photon fluorescent nanoprobe for monitoring intracellular nitric oxide levels

“…Overall, due to their reproducibility, stability, sensitivity, selectively, biological versatility and biocompatibility, DANPY-NO@AuNPs have proven to be an excellent tool to monitor and potentially quantify intracellular NO in vitro and their ability to be two-photon excitable makes them extremely valuable to aid our further understanding of the role that NO plays in biology, investigating 3D models and in vivo models. Although DANPY-NO@AuNPs exhibit a higher LOD than the recently published DANPY-NO molecular probe, 42 the nanoprobe exhibits a set of characteristics that make DANPY-NO@AuNPs excellent candidates for biomedical applications: (1) the broader dynamic range of the nanoprobe permits its application in biological samples with elevated concentrations of NO such as in the case of bacterial infection; (2) DANPY-NO@AuNPs have proven to be more stable in aqueous media compared to the molecular probe; and (3) importantly, having a nanoplatform permits the incorporation of other ligands such as reference ligands to obtain ratiometric nanoprobes for the precise quantification of intracellular NO, or drugs yielding nanoplatforms for theranostic applications.…”

“…1(a)), consists of a thiolated NO-sensitive ligand (DANPY-NO-ligand) self-assembled onto the surface of AuNPs. The fluorescent NO-sensitive ligand is a derivative of the recently published DANPY-NO 42 and, as such, presents an o -phenylenediamine linked to a naphthalimide core, which is responsible for the fluorescence quenching, due to a photoinduced electron transfer (PET), in the absence of NO. Upon reaction with NO in the presence of oxygen, and the consequent formation of the benzotriazole derivative, the PET is suppressed, the quenching effect is diminished, and the fluorescence emission of DANPY-NO-ligand is enhanced.…”

“…DANPY-NO was chosen as the NO-sensitive two-photon excitable fluorophore due to its high photostability, good sensitivity and excellent selectivity towards NO, its fluorescence stability at different biologically relevant pHs, its excellent biocompatibility and because it is extremely versatile in the detection of NO in cellular environments. 42 Furthermore, the versatile piperazine moiety in DANPY-NO allows the incorporation of a thiolated chain that can be linked onto the surface of AuNPs. The synthesis of DANPY-NO-ligand was accomplished in five steps starting from 4-bromo-1,8-naphthalic anhydride and 2-nitro- p -phenylenediamine (Scheme S1, ESI†) yielding the dimeric form of the thiolated monomer attached to the AuNPs.…”

“…Given the excellent performance of the two-photon excitable NO probe, DANPY-NO, recently reported by our group, 42 and our successful application of AuNPs-based systems for the detection and quantification of analytes of intracellular interest, 58,64,65 the work described here reports the first example of a two-photon nanoprobe based on AuNPs ( DANPY-NO@AuNPs , Fig. 1(a)) for the intracellular detection and potential quantification of NO that has been proven suitable to work in a broad range of cellular environments.…”

“…33–41 Amongst them, we recently reported a highly photostable and versatile two-photon fluorescent probe, DANPY-NO, for the detection of a wide range of intracellular NO concentrations in macrophages and endothelial cells using confocal laser scanning microscopy (CLSM), multiphoton microscopy, and flow cytometry. 42…”