Three enhanced fluorescence probes based on Rhodamine B-Schiff base structure were synthesized for detecting Cu. The corresponding detection limits were found to be 0.25 μM, 0.15 μM and 0.18 μM. Binding ratio and binding sites were determined by Job's and nuclear magnetic titration experiments. The binding constants obtained by the Benesi-Hildebrand equation to be 341.0 M,1.8 × 10 M, and 265.4 M, respectively. As isomers, the different effects of probes on Cu detection were researched. By adjusting the position and the size of the substituent group, the effects of binding sites and steric hindrance on the complexation ratio, response time and detection limit were discussed. Optimal spatial combination structure with Cu was obtained through energy calculation. Detection mechanism of Rhodamine B ring opening based on the complex of the Schiff base with Cu was confirmed. E. coli staining and detection of real water samples had expanded their applications.
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