Two-Photon Graphene Oxide/Aptamer Nanosensing Conjugate for <i>In Vitro</i> or <i>In Vivo</i> Molecular Probing

Two-photon excitation (TPE) with near-infrared (NIR) photons as the excitation source have the unique properties of lower tissue autofluorescence and self-absorption, reduced photodamage and photobleaching, higher spatial resolution, and deeper penetration depth (>500 μm). Carbon nanomaterials, for example, graphene oxide (GO), have the advantages of good biocompatibility, efficient transporters into cells, protecting the carried DNA or peptides from enzymatic cleavage, and super fluorescence quenching efficiency. By combination of the nanostructured carbon materials with the TPE technique, herein we have designed an aptamer-two-photon dye (TPdye)/GO TPE fluorescent nanosensing conjugate for molecular probing in biological fluids, living cells, and zebrafish. This approach takes advantage of the exceptional quenching capability of GO for the proximate TP dyes and the higher affinity of single-stranded DNA on GO than the aptamer–target complex. Successful <i>in vitro</i> and <i>in vivo</i> detection of ATP was demonstrated with this sensing strategy. Our results reveal that the GO/Aptamer–TPdye system not only is a robust, sensitive, and selective sensor for quantitative detection of ATP in the complex biological environment but also can be efficiently delivered into live cells or tissues and act as a “signal-on” <i>in vivo</i> sensor for specific, high-contrast imaging of target biomolecules. Our design provides a methodology model scheme for development of future carbon nanomaterial-based two-photon fluorescent probes for <i>in vitro</i> or <i>in vivo</i> determination of biological or biologically relevant species.