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 MnO nanosheets (MnO NSs) and alkaline phosphatase (ALP) was proposed. In this RF platform, the versatile MnO 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 MnO NSs into Mn, resulting in the fluorescence of AR decrease but fluorescence of CD recovery simultaneously. The regulation of MnO NSs for the fluorescence intensity of AR ( at 585 nm) and CDs ( 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 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, 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.