Single probe for imaging and biosensing of pH, Cu²⁺ ions, and pH/Cu²⁺ in live cells with ratiometric fluorescence signals

It is very essential to disentangle the complicated inter-relationship between pH and Cu in the signal transduction and homeostasis. To this end, reporters that can display distinct signals to pH and Cu are highly valuable. Unfortunately, there is still no report on the development of biosensors that can simultaneously respond to pH and Cu²⁺, to the best of our knowledge. In this work, we developed a single fluorescent probe, AuNC@FITC@DEAC (AuNC, gold cluster; FITC, fluorescein isothiocyanate; DEAC, 7-diethylaminocoumarin-3-carboxylic acid), for biosensing of pH, Cu²⁺, and pH/Cu²⁺ with different ratiometric fluorescent signals. First, 2,2′,2-(2,2′,2-nitrilotris(ethane-2,1-diyl)tris((pyridin-2-yl-methyl)azanediyl))triethanethiol (TPAASH) was designed for specific recognition of Cu²⁺, as well as for organic ligand to synthesize fluorescent AuNCs. Then, pH-sensitive molecule, FITC emitting at 518 nm, and inner reference molecule, DEAC with emission peak at 472 nm, were simultaneously conjugated on the surface of AuNCs emitting at 722 nm, thus, constructing a single fluorescent probe, AuNC@FITC@DEAC, to sensing pH, Cu²⁺, and pH/Cu²⁺ excited by 405 nm light. The developed probe exhibited high selectivity and accuracy for independent determination of pH and Cu²⁺ against reactive oxygen species (ROS), other metal ions, amino acids, and even copper-containing proteins. The AuNC-based inorganic-organic probe with good cell-permeability and high biocompatibility was eventually applied in monitoring both pH and Cu²⁺ and in understanding the interplaying roles of Cu²⁺ and pH in live cells by ratiometric multicolor fluorescent imaging.