Multiplex detection of nucleases by a graphene-based platform

In this article, we present a new method for the multiplex detection of nucleases by using graphene oxide (GO) as a platform. We introduce a Y-shaped DNA (Y-DNA) as the multiplex probe. The 5' termini of the Y-DNA are labeled with carboxy fluorescein (FAM), 6-carboxy-X-rhodamine (ROX) and cyanine 5 (Cy5) and they include three nuclease cleavage sites corresponding to PvuII, EcoRV and HaeIII, respectively. Upon the addition of nucleases, the nucleases cleave the corresponding sites in Y-DNA. Then, short dsDNA fragments containing fluorophores were released from the Y-DNA. These dsDNA fragments were unstable and easy to unwind into two short ssDNAs. They were then adsorbed onto the GO surface. Because of the excellent electronic transference of GO, the fluorescence intensity of the fluorophores can be quenched efficiently. Therefore, by monitoring the fluorophores' fluorescence change before and after the addition of the nucleases, it is easy to establish a platform of a Y-DNA/GO complex for the simultaneous multiplex detection of nucleases.