"Three-in-one" nanocomposites as multifunctional nanozymes for ultrasensitive ratiometric fluorescence detection of alkaline phosphatase.

Nanozymes, as a unique class of nanomaterials with enzyme-like properties, have attracted significant interest due to their potential applications in many significant fields. Great endeavours have been made to improve the catalytic activities of nanozymes; however, it is still a challenging issue to develop nanozymes that can functionally mimic multiplex enzymes with broader application prospects. Here, we develop a simple hydrothermal method to construct "three-in-one" nanocomposites as multifunctional nanozymes for the ultrasensitive ratiometric fluorescence detection of alkaline phosphatase (ALP). The prepared flower-like FeO nanocomposites (Fef NCs) are composed of ternary components, in which hierarchical MnO nanosheets (NSs) are assembled on FeO nanoparticles (NPs), followed by the decoration of CeO NPs. Fef NCs present tetra-enzyme-like activities, , oxidase-, peroxidase-, catalase-, and superoxide dismutase-like activity. More importantly, Fef NCs can effectively catalyze the oxidation of phenolic compounds (, 3,5-DTBC and dopamine) to produce the corresponding o-quinones, demonstrating specific catechol oxidase-like activity. Based on the excellent catalytic oxidation and fluorescence quenching abilities of Fef NCs, we established a ratiometric fluorescence strategy using two fluorogenic substrates for label-free, ultrasensitive, and selective detection of ALP. The fluorescence bioassay exhibits a linear relationship between the fluorescence ratio and the ALP concentration ranging from 0.2 to 1.0 mU mL, with a detection limit down to be 0.19 mU mL. Furthermore, this bioassay can detect ALP in mixture and human serum samples, presenting good selectivity as well as real-world applicability. This work not only provides a novel approach for the preparation of a multiple-enzyme-like nanozyme but also offers an advanced ratiometric fluorescence sensing platform for ultrasensitive bioanalysis.