Fluorogenic "on-off" nanosensor based on dual-quenching effect for imaging intracellular metabolite of various microalgae

Sensing intracellular compounds such as ATP in living microalgal cells is of great importance in diverse fields. To achieve this, nanosensing platform composed of graphene oxide (GO) and ATP aptamer (APT) was applied to diverse microalgal cells (Chlamydomonas reinhardtii, Chlorella vulgaris, Anabaena flos-aquae, and Ochromonas danica). The nanosized GO was characterized with atomic force microscopy (AFM), transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). The nanosensing platform (GO-APT) was prepared by attaching fluorophore-labeled APT to GO. GO-APT was applicable to only cell wall-deficient species (O. danica and mutant strains of C. reinhardtii) and the existence of flagella did not affect the uptake of the GO-APT by the cells. These results indicate that the cell wall is the primary barrier of GO-APT internalization for sensing application. To reduce the background fluorescence signal elicited by nonspecific displacement of the fluorophore-labeled probe, APT was modified as molecular beacon (MB) type (APTMB). Owing to the double quenching effect (by GO and quencher-labeled complementary sequence), the background signal significantly reduced. Cytotoxicity of GO-APTMB on the microalgal species was also tested. The application of GO-APTMB had no effect on the growth of microalgae. Given that diverse aptamer sequences had been screened, the sensing platform is not limited for detecting ATP only, but also can be applied to other metabolite imaging by simply changing the aptamer sequences. Our research will contribute to broadening the application of GO and aptamer beacon complex for intracellular metabolite imaging and detecting.