Phosphate-perylene modified G-quadruplex probes for the detection of Pb²⁺ using fluorescence anisotropy

A simple and universal phosphate-perylene modification strategy was applied in the development of G-quadruplex probes with thrombin binding aptamer (TBA) and [d(TGGGT)₄] (G4) sequences. A perylene moiety was inserted at different phosphate positions of oligonucleotides without a significant effect on the G-quadruplex structures. Upon binding with K⁺ or Pb²⁺, these probes showed different perylene fluorescence anisotropy responses due to the different labeling positions and G-quadruplex structures. Two probes (G4-9 and TBA-9) were successfully used in Pb²⁺ detection through fluorescence anisotropy. Once the complexes of Pb²⁺ with G4-9 or TBA-9 were formed, the rotational diffusion of the perylene moiety was limited, resulting in a significant increase in fluorescence anisotropy. Both probes showed good sensitivity to Pb²⁺ and their fluorescence anisotropy signals demonstrated good linear responses to the logarithm of Pb²⁺ concentrations and the detection limits were 24.5 nM and 30.0 nM for TBA-9 and G4-9, respectively.