Dual-color encoded DNAzyme nanostructures for multiplexed detection of intracellular metal ions in living cells

The detection of intracellular metal ions is of great importance in understanding metal homeostasis in cells and related diseases, and yet it remains a significant challenge to achieve this goal. Based on a new self-assembled and dual-color encoded DNAzyme nanostructure, we describe here an approach for multiplexed sensing of UO22+ and Pb2+ in living cells. The fluorescently quenched nanoprobes can be prepared by simple thermal annealing of four ssDNAs containing the metal ion-dependent enzymatic and substrate sequences. The self-assembly formation of the nanostructures are verified by native polyacrylamide gel electrophoresis. The target metal ions can cleave the substrate sequences in the DNAzyme nanostructures to recover fluorescent emissions at different wavelengths for sensitive and selective in vitro multiplexed detection of UO22+ and Pb2+ with the detection limits of 0.6 nM and 3.9 nM, respectively. Importantly, we demonstrate that these nanoprobes are stable in cell lysates and can enter cells without the aid of any transfection agents for simultaneous imaging intracellular UO22+ and Pb2+. Moreover, the nanoprobes offer excellent biocompatibility and non-cytotoxicity. With these unique features, the dual-color encoded nanostructures presented here can thus offer new opportunities for multiplexed detection of specific intracellular species.