Ratiometric fluorescence determination of alkaline phosphatase activity based on carbon dots and Ce³⁺-crosslinked copper nanoclusters

A new ratiometric fluorescent probe for efficient determination of ALP was developed. The probe was constructed by combining Ce³⁺-crosslinked copper nanoclusters (Ce³⁺-CuNCs) which exhibit the aggregation-induced emission (AIE) feature with carbon dots (CDs). The introduction of phosphate (Pi) induced the generation of CePO₄ precipitation, resulting in significant decrease of fluorescence emission of CuNCs at 634 nm. At the same time, the fluorescence of CDs at 455 nm was obviously enhanced, thus generating ratiometric fluorescence response. Based on the fact that the hydrolysis of pyrophosphate (PPi) by ALP can produce Pi, the CD/Ce³⁺-CuNCs ratiometric probe was successfully used to determine ALP. A good linear relationship between the ratiometric value of F₄₅₅/F₆₃₄ and ALP concentrations ranging from 0.2 to 80 U·L^{− 1} was obtained, with a low detection limit of 0.1 U·L^{− 1}. The ratiometric responses of the probe resulted in the visible fluorescence color change from orange red to blue with the increase of ALP concentration. The smartphone-based RGB recognition of the fluorescent sample images was used for ALP quantitative determination. Graphical abstract