A cooperative sequential binding rhodamine-based fluorescent probe for total copper sensing: From environmental...

Copper is an essential trace element involved in various biological processes. However, disruption of copper homeostasis can have adverse effects on human health. Selective and sensitive detection of copper ions in both oxidation state (Cu²⁺ or Cu⁺) is therefore important, as Cu²⁺ is toxic at elevated levels, while intracellular Cu⁺ levels are often elevated in cancer cells. Because Cu²⁺ and Cu⁺ predominate in extracellular (oxidizing) and intracellular (reducing) environments, respectively, a single probe capable of detecting both species would be particularly valuable.

Here, we present a rhodamine B hydrazide-based fluorescent probe (probe 1) for the selective detection of both Cu²⁺ and Cu⁺. Probe 1 exhibited good selectivity and sensitivity toward Cu²⁺ in a H₂O/acetonitrile (1:1, v/v) mixture, with a limit of detection (LOD) of 2.2 nM, and toward Cu⁺ in aqueous solution, with an LOD of 12 nM. The probe enabled sensitive detection and quantification of Cu²⁺ in beverages and environmental water samples and selectively detected intracellular Cu⁺, allowing differentiation between cancerous and normal cells. Interestingly, initial Cu⁺ binding generates the secondary metal-binding site, resulting in cooperative sequential binding. Subsequent metal binding induces conformational rigidification, enhancing fluorescence intensity and stability under strongly basic conditions.

The spatial coupling between the two metal-binding sites enables communication between successive binding events, demonstrating how cooperative sequential binding can enhance sensing performance. This study highlights a useful molecular design strategy for developing multifunctional fluorescent probes capable of responding to different oxidation states of the same metal ion. These findings demonstrate the potential of exploiting cooperative binding interactions in small-molecule fluorescent probes to improve sensing capabilities and broaden their applications in environmental analysis and cellular imaging.