A novel strategy for Escherichia coli detection in raw beef in combination with click chemistry

Bacteria-assisted click chemistry for enhanced nanozyme-based Escherichia coli (E. coli) detection. The size and shape of the alkyne-modified Fe₃O₄@Au NPs were 110 nm and urchin-like, respectively. The azide-functionalized Au QDs were spherical in shape, and their size was ~5 nm. The absorbance and emission peaks of Fe₃O₄@Au NPs and Au QDs were located at 526 nm and 520 nm, respectively. The nanozymatic activity of both materials significantly improved in the presence of Cu²⁺, E. coli, and 3,3',5,5'-tetramethylbenzidine (TMBZ)/H₂O₂ mixture. Here, the bacterial metabolic product Cu⁺ was utilized to trigger the click reaction between alkyne-modified Fe₃O₄@Au NPs and azide-functionalized Au QDs which enables magnetic separation. The calculated limits of detection (LODs) of E. coli were as high as 5 CFU/mL and 25 CFU/mL in the PBS buffer and spiked raw meat samples, respectively. The proposed nanozymatic E. coli assay was selective in the presence of other bacteria.