In situ simultaneous monitoring of ATP and GTP using a graphene oxide nanosheet-based sensing platform in living cells.

Here we present a detailed protocol for in situ multiple fluorescence monitoring of adenosine-5'-triphosphate (ATP) and guanosine-5'-triphosphate (GTP) in MCF-7 breast cancer cells by using graphene oxide nanosheet (GO-nS) and DNA/RNA aptamers. FAM-labeled ATP aptamer and Cy5-modified GTP aptamer are used to construct the multiple aptamer/GO-nS sensing platform through 'π-π stacking' between aptamers and GO-nS. Binding of aptamers to GO-nS guarantees the fluorescence resonance energy transfer between fluorophores and GO-nS, resulting in 'fluorescence off'. When the aptamer/GO-nS are transported inside the cells via endocytosis, the conformation of the aptamers will change on interaction with cellular ATP and GTP. On the basis of the fluorescence 'off/on' switching, simultaneous sensing and imaging of ATP and GTP in vitro and in situ have been realized through fluorescence and confocal microscopy techniques. In this protocol, we describe the synthesis of GO and GO-nS, preparation of aptamer/GO-nS platform, in vitro detection of ATP and GTP, and how to use this platform to realize intracellular ATP and GTP imaging in cultured MCF-7 cells. The preparation of GO-nS is anticipated to take 7-14 d, and assays involving microscopy imaging and MCF-7 cells culturing can be performed in 2-3 d.