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

Abstract

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 in vitro and in vivo 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" in vivo 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 in vitro or in vivo determination of biological or biologically relevant species.

Fluorescence and Sensing Applications of Graphene Oxide and Graphene Quantum Dots: A Review

“…In addition, GO as a FRET quencher has also been used for in vitro and in vivo molecular probing based on two-photon excitation spectroscopy. [34] By replacing the capture and detection reagents, the GO sensor platform has been extended for the detection of proteins, DNA, drugs, heavy metals, et al [33b, 35] …”

Intracellular Detection of ATP Using an Aptamer Beacon Covalently Linked to Graphene Oxide Resisting Nonspecific Probe Displacement

“…Intracellular adenosine triphosphate (ATP) detection using aptamer/GO complexes have been demonstrated by a few groups. [38][39][40][41][42] A potential problem of the physisorption system is that the adsorbed probe might be displaced by non-target molecules, thus producing false positive signals. This is particularly problematic for intracellular detection since a high concentration of proteins and nucleic acids exist in both the culture medium and in cells.…”

Aptamer-based nanobiosensors

“…More recently, AuNPs have been widely used in the fluorometric aptasensors based on the superquenching property of AuNP for almost of fluorescence materials (Liu et al, 2014). The quenching effect of AuNP represents orders of magnitude higher than one of other organic quenching molecules (Fan et al, 2003;Liu et al, 2007a).…”

“…By the addition of targets, then the aptamers were displaced from GO surface by target induced conformational change of aptamers to folded structure, resulting the increasing of fluorescence signal. This assay was applied to not only in vitro analysis, but also in vivo molecular probing of ATP (Yi et al, 2014). Instead of fluorescence dyes, QD was also used for GO-based FRET aptasensors (Zhou et al, 2014).…”