Abstracts

The novel bioluminescent fusion protein, which was formed by the green-blue emitting Amydetes vivianii firefly luciferase (Amy-WT) linked with the N-terminal ZZ portion of protein A (chimeric ZZ-Amy), has been efficiently applied in immun blots for detection of SARS-CoV-2 nucleoprotein. The chimeric ZZ-Amy displays a slightly higher and more sustained luminescent signal when compared to commercial cheiluminescent HRP-labeled secondary antibodies. The increase of Pyrearinus termitilluminans (PyLuc) and Phrixotrix red emitting (RELuc) luciferases stability when associated with gold nanoparticles (AuNPs) capped with dithiol polyethylene glycol (PEG(SH)₂AuNPs) inspired similar Nano/Bio construction for Amy-WT and chimeric ZZ-Amy luciferases. The association of the enzymes with the PEG(SH)₂AuNPs did not affect the enzymatic structure, as attested by FTIR analysis and bioluminescence spectra. Scanning electron microscopy images showed 10 nm AuNPs aggregated in a dense PEG(SH)₂ corona that became rarefied in the presence of Amy-WT. Contrary to the observed for PyLuc and RELuc, Amy-WT and chimeric ZZ-Amy associated with PEG(SH)₂AuNPs exhibited a significant luminescence increase (absolute activity) with a slight stability increase. The spectral overlap of luciferases with the absorption band of the 10 nm AuNPs maximizes the effect of confined plasmons on the optical density of states near the emission peaks of Amy-WT and ZZ-Amy. The Nano/ZZ-Amy construction improves the detection limits of the bioluminescence for several practical applications.