Capillary electrophoresis with dual detection UV/C4D for monitoring myrosinase-mediated hydrolysis of thiol glucosinolate designed for gold nanoparticle conjugation

Abstract

Capillary electrophoresis (CE) with dual UV and conductivity detection was used for the first time to monitor the functionalization of gold nanoparticles (AuNPs), a process catalyzed by an enzyme, myrosinase (Myr). A thiol glucosinolate (GL-SH) designed by our group was used as substrate.Hydrolysis of free and immobilized GL-SH was characterized using off-line and on-line CE-based enzymatic assays. The developed approaches were validated using sinigrin, a well-referenced substrate of Myr. Michaelis-Menten constant of the synthetized GL-SH was comparable to sinigrin, showing that they both have similar affinity towards Myr. It was demonstrated that transverse diffusion of laminar flow profiles was well adapted for in-capillary Mixing of nanoparticles (AuNPs) with proteins (Myr) provided that the incubation time is inferior to 20 min. Only low reaction volume (nL to few μL) and short analysis time (<5 min) were required. The electrophoretic conditions were optimized in order to evaluate and to confirm the AuNPs stability before and after functionalization by CE/UV based on surface plasmon resonance band red-shifting. The hydrolysis of the functionalized AuNPs was subsequently evaluated using the developed CE-C4D/UV approach. Repeatabilities of enzymatic assays, of electrophoretic analyses and of batch-to-batch functionalized AuNPs were excellent.