Real-Time Electrochemical Measurement of N-Acetyl-ß-D-Glucosaminidase Activity, Using Redox-Tagged N-Acetyl-ß-D-Glucosaminide Hydrolysis as Proof-of-Principle

In-situ amperometry was used as a convenient non-optical tool for the assessment of the chitooligosaccharide-hydrolyzing activity of N-acetyl-$\beta $ -D- glucosaminidase from the marine Vibrio harveyi (VhGlcNAcase). Signal generation used a 3-mm-diameter boron-doped diamond (BDD) electrode for the anodic oxidation of 4-methylumbelliferone (4MU), which is released from the model substrate 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (GlcNAc-4MU) by enzymic hydrolysis. The recorded rise in 4MU electrode current reported the release of the redox label in real time and amperometric data analysis reproduced the Michaelis-Menten VhGlcNAcase activity plots derived from standard spectroscopic assays. Since it provides continuous readout, amperometry avoids the use of reaction-stopping chemicals and is also practical in turbid test solutions. Furthermore, the established electrochemical enzyme activity assay offers the chance to study novel substrate-attached redox tags and to explore assay miniaturization through implementation in microfluidic devices, enables portable assay development through the use of mini-potentiostats with smart phone/tablet connectivity and aids assay automation through combination with platforms for robotic electrochemistry.