Nanozyme–Enzyme Cascade Reaction-Enhanced Ratiometric Fluorescence Immunosensing Platform for Sensitive and Accurate Detection of Ractopamine

As the most widely used immunoassay, enzyme-linked immunosorbent assay (ELISA) relies on perishable enzymes and usually provides poor sensitivity and stability, limiting its application in detecting trace analytes in harsh environments. Herein, a new ratiometric fluorescence (RF) sensing platform enhanced by a nanozyme–enzyme cascade reaction composed of MnO₂ nanosheets (MnO₂ NSs) and alkaline phosphatase (ALP) was proposed. In this RF platform, the versatile MnO₂ NSs worked as a robust oxidase-like nanozyme to catalyze nonfluorescent Amplex Red (AR) into fluorescent resorufin and as a quencher to quench the fluorescence of carbon dots (CDs). Given that ALP could catalyze ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA), followed by AA reducing MnO₂ NSs into Mn²⁺, resulting in the fluorescence of AR decrease but fluorescence of CD recovery simultaneously. The regulation of MnO₂ NSs for the fluorescence intensity of AR (E_m at 585 nm) and CDs (E_m at 460 nm) could cascade with ALP-triggered reaction to construct an RF sensing platform based on RF signal (F585/F460) output. The proposed RF sensing platform could achieve a superior limit of detection (LOD) of 0.037 mU mL^–1 for ALP activity quantification. Inspired by the excellent performance of the RF sensing platform, a sensitive and accurate RF ELISA for ractopamine (RAC) detection was established with an LOD of 0.029 ng mL^–1, which was nearly 5.5 times more sensitive than traditional colorimetric ELISA. This RF sensing platform based on robust nanozyme-triggered enzymatic cascade signal amplification and excellent RF signal readout has offered a powerful and universal platform for RAC and other trace target detection.