Two-Photon Ratiometric Fluorescent Sensor Based on Specific Biomolecular Recognition for Selective and Sensitive Detection of Copper Ions in Live Cells

In this work, we develop a ratiometric two-photon fluorescent probe, ATD@QD-E2Zn2SOD (ATD = amino triphenylaminedendron, QD = CdSe/ZnSe quantum dot, E2Zn2SOD = Cu-free derivative of bovine liver copper–zinc superoxide dismutase), for imaging and sensing the changes of intracellular Cu2+ level with clear red-to-yellow color change based on specific biomolecular recognition of E2Zn2SOD for Cu2+ ion. The inorganic–organic nanohybrided fluorescent probe features two independent emission peaks located at 515 nm for ATD and 650 nm for QDs, respectively, under two-photon excitation at 800 nm. Upon addition of Cu2+ ions, the red fluorescence of QDs drastically quenches, while the green emission from ATD stays constant and serves as a reference signal, thus resulting in the ratiometric detection of Cu2+ with high accuracy by two-photon microscopy (TPM). The present probe shows high sensivity, broad linear range (10–7–10–3 M), low detection limit down to ∼10 nM, and excellent selectivity over other metal ions, ami...