Indicator-Free Detection of Hyaluronidase Based on a Conductivity-Regulated Bipolar Electrochemiluminescence Platform

Hyaluronidase (HAase) detection plays a critical role in cancer diagnosis and treatment. Most HAase biosensors require the incorporation of exogenous indicators into the sensing reaction for signal transduction, which might involve complex synthesis and modification procedures or potentially interfere with enzymatic activity. Herein, based on the fact that the electrochemiluminescence (ECL) can be regulated via a bipolar electrode (BPE) through the alteration of conductivity in the enzymatic reaction solution, we develop an indicator-free HAase biosensor based on the bipolar-based ECL (BP-ECL) platform. HAase degrades negatively charged macromolecular hyaluronic acid (HA) into small anionic fragments, thereby increasing the conductivity of the sensing solution. As a result, the variation of conductivity adjusts the voltage drop across the BPE and promotes electron transfer reactions at the anodic pole, thereby enhancing the ECL emission in the reporting cell. The HAase activity can be quantified by monitoring the ECL response modulation in the BP-ECL sensing platform. Given the optimal experimental conditions, the proposed BP-ECL biosensor showed a good linear relationship with the concentration of HAase in the range of 1∼60 U/mL and a detection limit of 0.13 U/mL. The developed HAase biosensor eliminates the need for exogenous indicators in bioreaction solutions and has been successfully applied for target detection in real samples with satisfactory performance.