Sensitive ratiometric fluorescence probe based on chitosan carbon dots and calcein for Alkaline phosphatase detection and bioimaging in cancer cells

Alkaline phosphatase (ALP) is a commonly used marker in clinical practice, and this enzyme is a key indicator for diagnosing various diseases. In this study, we describe the development of a reliable and novel fluorescent assay for ALP detection based on chitosan carbon dots (C-CDs, peak emission, 412 nm) and calcein (peak emission, 512 nm). In the presence of Eu³⁺ (which binds calcein), the fluorescence intensity of calcein is quenched. Utilizing the ALP-triggered generation of phosphate ions (PO₄³⁻) from the substrate p-nitrophenyl phosphate (pNPP), the Eu³⁺ ions bind PO₄³⁻ (which shows a higher affinity toward Eu³⁺ than calcein), and the fluorescence of calcein is recovered. As a consequence, C-CDs fluorescence is decreased by inner filter effect (IFE). Exploiting these changes in the fluorescence intensity ratio of C-CDs and calcein, we developed a high sensitivity, accurate, and easily synthesized ratiometric fluorescence probe. Our novel fluorescent bioassay demonstrates good linear relationship in the 0.09–0.8 mU mL⁻¹ range, with a low detection limit of 0.013 mU mL⁻¹. The excellent applicability of this novel assay in HepG2 cells and human serum samples demonstrates that our novel method has excellent biomedical research and disease diagnosis prospects.