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

Abstract

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 Fe₃O₄ nanocomposites (Fef NCs) are composed of ternary components, in which hierarchical MnO₂ nanosheets (NSs) are assembled on Fe₃O₄ nanoparticles (NPs), followed by the decoration of CeO₂ NPs. Fef NCs present tetra-enzyme-like activities, i.e., oxidase-, peroxidase-, catalase-, and superoxide dismutase-like activity. More importantly, Fef NCs can effectively catalyze the oxidation of phenolic compounds (i.e., 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⁻¹, with a detection limit down to be 0.19 mU mL⁻¹. 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.