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 Eu (which binds calcein), the fluorescence intensity of calcein is quenched. Utilizing the ALP-triggered generation of phosphate ions (PO) from the substrate p-nitrophenyl phosphate (pNPP), the Eu ions bind PO (which shows a higher affinity toward Eu 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 range, with a low detection limit of 0.013 mU mL. 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.