Electrochemiluminescence biosensor for hyaluronidase activity detection and inhibitor assay based on the electrostatic interaction between hyaluronic acid and Ru(bpy)32+

The development of simple and sensitive method for hyaluronidase (HAase) detection is significant for the clinical diagnosis and treatment of cancer as HAase is a potential tumor marker. In this study, a simple but sensitive electrochemiluminescence (ECL) biosensor has been designed for HAase detection based on the electrostatic interaction of anionic hyaluronic acid (HA) and cationic luminous reagent Ru(bpy)32+. Ru(bpy)32+ is adsorbed on HA through electrostatic interaction to form HA-Ru(bpy)32+ complex. The Ru(bpy)32+ in the HA-Ru(bpy)32+ complex cannot be filtered out because of its large molecular weight. In the presence of HAase, the HA in HA-Ru(bpy)32+ can be cleaved into fragments by enzymatic hydrolysis. As a result, the Ru(bpy)32+ attached on the HA fragments can be separated from the HA-Ru(bpy)32+ complex by centrifugation easily. The resulting ultrafiltrate containing Ru(bpy)32+ can be used to characterize the concentration of HAase in the sample. The ECL intensity of Ru(bpy)32+ has a linear relationship with the concentration of HAase in the range of 2.0-40 U/mL with a detection limit of 0.33 U/mL. The proposed ECL system has been applied to detect HAase in urine samples with satisfied results and HAase inhibitor with high efficiency.