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

摘要

In this work, we develop a ratiometric two-photon fluorescent probe, ATD@QD-E_2Zn_2SOD (ATD = amino triphenylamine dendron, QD = CdSe/ZnSe quantum dot, E_2Zn_2SOD = Cu-free derivative of bovine liver copper-zinc superoxide dismutase), for imaging and sensing the changes of intracellular Cu~(2+) level with clear red-to-yellow color change based on specific biomolecular recognition of E_2Zn_2SOD for Cu~(2+) 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 Cu~(2+) 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 Cu~(2+) 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 Cu~(2+) 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 Cu~(2+) in live cells.