Results and Troubleshooting in Intracellular Heavy Metal-Targeting of a Fluorescent Hydroxy Naphthyl-Methylbenzoxazole-Based Dye

Background: The increasing concern regarding heavy metal contamination has turned their selective detection into a critical branch of environmental and biomedical research. Among various pollutants, Cadmium (Cd²⁺) is particularly hazardous; classified as a Class I carcinogen, it accumulates through the food chain, leading to severe organ damage and ecotoxicological impacts. Conventional instrumental techniques, such as atomic absorption spectrometry, cannot analyze trace metal ions in still living cells. To bridge this gap, fluorescent small molecules have emerged as ideal tools for real-time, non-destructive monitoring of intracellular metal fluctuations. Materials and Methods: The efficiency of a recently developed 2-(2-hydroxynaphthyl)benzoxazole-based probe (HNBO-DEN, where DEN represents the diethylenetriamine chelating unit) was evaluated. Testing was performed on human colorectal adenocarcinoma cells (HT-29) and hemocytes from the marine bioindicator Mytilus galloprovincialis. Flow Cytometry and Confocal Microscopy were utilized to assess probe internalization, Cd²⁺ detection efficiency, and cell viability through 7-AAD staining. Results: In biological models, both systems without significant cytotoxicity at working concentrations, identified cadmium. HNBO-DEN appeared to have good sensitivity to detect cytoplasmic Cd²⁺. In the HT-29 cells, a significant increase in mean fluorescence intensity was observed following 24 h of exposure to 150 μM CdCl₂. In mussel hemocytes, granulocytes proved to be the most sensitive subpopulation for tracing environmental Cd²⁺ concentrations (from 1 nM to 1 mM), showing a proportionality between metal concentration and fluorescence intensity. The study also addressed troubleshooting related to dye specificity, cell autofluorescence and the accurate distinction between viable and damaged cells, crucial for environmental assessments. Conclusion: The free fluorescent probe provides an effective diagnostic tool for heavy metal detection, mapping the presence of the metal in the cytosol. Further investigations will focus on the detection and actions of Cadmium as a metallo-estrogen in breast cancer cell models, such as MCF7.