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 triphenylamine dendron, 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 E₂Zn ₂SOD 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, amino acids, and other biological species. Meanwhile, a QD-based inorganic-organic probe demonstrates long-term photostability, good cell-permeability, and low cytotoxicity. As a result, the present probe can visualize Cu²⁺ changes in live cells by TPM. To the best of our knowledge, this is the first report for the development of a QD-based two-photon ratiometric fluorescence probe suitable for detection of Cu²⁺ in live cells.