Ratiometric fluorescent sensing of pH values in living cells by dual-fluorophore-labeled i-motif nanoprobes.

We designed a new ratiometric fluorescent nanoprobe for sensing pH values in living cells. Briefly, the nanoprobe consists of a gold nanoparticle (AuNP), short single-stranded oligonucleotides, and dual-fluorophore-labeled i-motif sequences. The short oligonucleotides are designed to bind with the i-motif sequences and immobilized on the AuNP surface via Au-S bond. At neutral pH, the dual fluorophores are separated, resulting in very low fluorescence resonance energy transfer (FRET) efficiency. At acidic pH, the i-motif strands fold into a quadruplex structure and leave the AuNP, bringing the dual fluorophores into close proximity, resulting in high FRET efficiency, which could be used as a signal for pH sensing. The nanoprobe possesses abilities of cellular transfection, enzymatic protection, fast response and quantitative pH detection. The in vitro and intracellular applications of the nanoprobe were demonstrated, which showed excellent response in the physiological pH range. Furthermore, our experimental results suggested that the nanoprobe showed excellent spatial and temporal resolution in living cells. We think that the ratiometric sensing strategy could potentially be applied to create a variety of new multicolor sensors for intracellular detection.