Elongated Carbon Dots with Ratiometric Fluorescence for Biomedical pH Sensing

Herein, we report a hydrothermal strategy to synthesize Rhodamine B-conjugated carbon dots (CD-RhB) using milk powder as a carbon precursor. The resulting nanostructures exhibit an unprecedented elongated morphology, with an average length of 21.9 ± 6.1 nm, as revealed by high-resolution transmission electron microscopy. This shape is hypothesized to originate from the partial preservation of casein micellar subdomains, particularly cylindrical, water-separated nanostructures within micelles, under high-concentration (57.14 mg/mL milk powder; ∼18.3 mg/mL casein) and elevated-temperature (180 °C) synthesis conditions. The CD-RhB shows dual-emission behavior, enabling a ratiometric fluorescence response toward pH changes between pH 8 and pH 5.5, with a sensitivity of 27.74% change in emission ratio per unit change in pH. The limit of detection was found to be 0.5 pH units, demonstrating a 69.36% increase in fluorescence emission toward pH sensitivity. They remain unperturbed in the presence of metal ions, except for ferric (Fe³⁺) ions at concentrations exceeding 50 μM, where the CD-RhB exhibits a detection limit of 100 μM with reliable reproducibility, as evidenced by consistent fluorescence intensity ratios across triplicates. Notably, such elevated Fe³⁺ levels are found in pathological conditions, allowing CD-RhB to also function as dual sensors for both pH and Fe³⁺ in extreme environments. The nanoprobes demonstrate very good reversibility between pH 5 and 7 over multiple pH switching cycles and low cytotoxicity across a range of concentrations in HeLa cells. Our findings introduce unique morphology-driven optical behavior in carbon dots and highlight their potential for robust biosensing applications.