A Dual-Functional Rhodamine B and Azo-Salicylaldehyde Derivative for Simultaneous Detection of Copper and Hypochlorite: Synthesis, Biological Applications and Theoretical Insights

Abstract

A multifunctional rhodamine derivative containing azo-salicylaldehyde (BBS) was designed and synthesized as a colorimetric and fluorescence turn-on probe for the selective detection of the cation copper (Cu²⁺) and the anion hypochlorite (OCl¯) in aqueous media. In the presence of Cu²⁺, the probe BBS exhibited a turn-on absorption and fluorescence change at 554 nm and 585 nm, respectively. The binding mechanism of BBS with Cu²⁺ induces the opening of a spirolactam ring in the rhodamine moiety by the formation of metal-ligand complex, achieving 10-fold enhancement in fluorescence and quantum yield along with a binding constant of 1 × 10⁴ M⁻¹ and a detection limit of 2.61 μM. On addition of OCl¯ enhanced the absorbance and fluorescence intensities at 520 nm and 575 nm respectively. The probe BBS undergoes hypochlorite-mediated oxidation followed by hydrolysis, resulting the formation of rhodamine B itself that is detectable by a naked-eye color and fluorescence enhancement by 11-fold with a high quantum yield and a detection limit of 1.96 µM. For practical applications, sensor BBS can be used to detect Cu²⁺ in water analysis and on cotton swabs. For biological applications, the interaction of the BBS-Cu (II) complex with transport proteins like bovine serum albumin (BSA) and ct-DNA was investigated through UV-vis and fluorescence titration experiments. Additionally, the structural optimization of BBS and BBS-Cu (II) complex was demonstrated using DFT, and the interactions of the BBS-Cu(II) complex with BSA and ct-DNA were analysed through theoretical docking studies. Bioimaging studies were conducted by capturing fluorescence images of BBS with Cu²⁺ and OCl¯ in a physiological medium containing living plant tissue using green gram seeds.