Ratiometric fluorescence immunoassay based on PtPdRu trimetallic nanozyme with enhanced oxidase-like activity for sensitive detection of xylazine.

Xylazine (XYL), an illicit opioid adulterant, its abuse and misuse can cause serious food safety incidents, drug-facilitated crimes, and even death. Therefore, to address the urgent need for XYL monitoring, a novel ratiometric fluorescence (RF) nanozyme-linked immunosorbent assay (NLISA) was proposed. This RF-NLISA used a robust polyvinylpyrrolidone ligand assisted trimetallic PtPdRu nanocluster (NC) nanozyme with high oxidase-like activity as the signal label. The PtPdRu NC could simultaneously catalyze the dual substrates scopoletin and Amplex Red to induce opposing fluorescence intensity changes, enabling sensitive RF signal output. Based on PtPdRu NC catalysis to trigger self-calibrating RF signals, the RF-NLISA achieved a limit detection of 0.0092 ng mL-1. This represents a nearly 10-fold sensitivity improvement over traditional horseradish peroxidase-based colorimetric ELISA, while maintaining excellent specificity and accuracy. This novel PtPdRu NC nanozyme facilitated RF-NLISA provides a sensitive and accurate sensing platform for XYL abuse monitoring in various food and human urine samples.