Synthesis of bioluminescent gold nanoparticle-luciferase hybrid systems for technological applications

Bioluminescent gold nanoparticles (AuNPs) were synthesized in situ using dithiol-terminated polyethylene glycol (PEG(SH)₂) as reducer and stabilizing agents. Hybrid Au/F₃O₄ nanoparticles were also produced in a variation of synthesis, and both types of nanostructures had the polymer capping replaced by L-cysteine (Cys). The four types of nanoparticles, PEG(SH)₂AuNPs, PEG(SH)₂Au/F₃O₄NPs, CysAuNPs, and CysAu/F₃O₄NPs were associated with purified recombinant Pyrearinus termitilluminans green emitting click beetle luciferase (PyLuc) and Phrixotrix hirtus (RELuc) red-emitting railroad worm luciferase. Enzyme association with PEG(SH)₂ was also investigated as a control. Luciferases were chosen because they catalyze bioluminescent reactions used in a wide range of bioanalytical applications, including ATP assays, gene reporting, high-throughput screening, bioluminescence imaging, biosensors and other bioluminescence-based assays. The immobilization of PyLuc and RELuc promoted partial suppression of the enzyme luminescence activity in a functionalization-dependent way. Association of PyLuc and RELuc with AuNPs increased the enzyme operational stability in relation to the free enzyme, as evidenced by the luminescence intensity from 0 to 7 h after substrate addition. The stability of the immobilized enzymes was also functionalization-dependent and the association with CysAuNPs was the condition that combined more sustained luminescent activity with a low degree of luminescence quenching. The higher enzymatic stability and sustained luminescence of luciferases associated with nanoparticles may improve the applicability of bioluminescence for bioimaging and biosensing purposes.