Covalent Coupling Assisted Hydrophilic Perovskite Spheres for Ratiometric Fluorescent Visual Multichannel Immunoassay

Abstract Quantitative fluorescence immunoassay is essential for the construction of biosensing mechanisms and the quantification of trace markers. But the interference problems caused by low fluorescence efficiency and broad fluorescence spectrum of fluorescent probes have hindered the continued development of ratiometric fluorescence sensing in biosensing. Perovskite materials, with ultra‐high color purity (FWHM < 30 nm) and photoluminescence quantum yield (PLQY) (close to 100%), are expected to be next‐generation fluorescent probes. However, poor water stability and biocompatibility are still non‐negligible in biosensor applications. In this work, hyperstatic perovskite fluorescent microspheres prepared by swelling‐shrinking method can be used as ratiometric fluorescence signals and biological immunoassay platforms. Meanwhile, inspired by p‐aminophenol (AP) controlled synthesis and the catalytic reaction of 4‐aminophenol phosphate (APP) triggered by alkaline phosphatase (ALP), a strategy to prepare fluorescent nanoparticles as fluorescence signals for ALP detection is proposed. Most importantly, it is proposed for the first time to combine this enzymatic fluorescence with perovskite materials using covalent linkage to create a novel cascade immunoassay and use it for quantitative and visualization determination of hepatitis B surface antigen (HBsAg) for application verification. These results indicate the biosensing potential of perovskite materials and provide a pathway for high sensitivity enzyme detection and enzyme triggered immune detection.