Using Electrochemical Impedance Spectroscopy to Study Biofilm Growth in a 3d-Printed Flow Cell System

Biofilm contamination is a widespread issue that can occur anywhere when organisms attach to surfaces in the presence of water. In industrial environments, formation of biofilms can lead to component failure, material degradation, and biofouling or spoilage, which collectively come with significant economic costs. Microfabricated electrochemical impedance spectroscopy (EIS) sensors have emerged as a promising tool for monitoring biofilm as EIS sensors capture information about biofilm growth autonomously in real-time; however, sensors suffer from drift, and the technique lacks temporal interpretation of dynamic biofilm processes. In this work, microfabricated sensors featuring gold micro-interdigitated electrodes (μIDEs) were modified with an electrically conductive polymer layer resulting in EIS measurement variability that was significantly reduced compared to unmodified sensors, and enabled highly stable, time-resolved EIS measurements. EIS characterization of Pseudomonas aeruginosa biofilm in parallel with high-resolution confocal laser scanning microscopy (CLSM) was performed using a novel 3D-printed flow cell system, resulting in distinct changes to EIS data corresponding with consistent biofilm growth. We have shown that EIS microsensors can detect four stages of biofilm: (i) initial biofilm attachment to the sensor substrate, (ii) early-stage irreversible biofilm proliferation characterized by sparse biofilm coverage, (iii) mature biofilm detection characterized by uniform biofilm coverage, and (iv) changes due to detachment and regrowth of biofilm.