Biofilm Spatial Monitoring By Multiple Electrochemical Techniques Integrated with Optical Microscopy and a Microfluidic Device
Authors: Belous Maruani Oriya , Milo Anat , Ben-Yoav Hadar 
Published: August 2024

Biofilm represents the inherent structure of bacterial populations, consisting of bacteria encapsulated within extracellular polymeric substances they secrete. The natural formation of a biofilm acts as a physical barrier, offering protection against environmental changes and increasing bacterial antibiotic resistance, often up to 1,000 times. To enhance our understanding of these intricate biosystems, electrochemical analytical micro-systems are crucial to mimic the biofilm’s behavior in nature and to monitor responses to diverse treatments. Current electrochemical micro-systems, however, face a fundamental limitation, employing a single electrode to characterize biofilm behavior [1], despite biofilms being inherently three-dimensional and non-uniformly distributed across the electrode. Here, we introduce a novel electrochemical micro-systems that is based on an array of microelectrodes and microfluidics to provide spatial monitoring of the biofilm. Fabricated through conventional photolithography and lift-off techniques, the micro-system comprises five individual gold microelectrode arrays covered with a polydimethylsiloxane microfluidic channel (Fig. 1A) and linked to a reference Ag/AgCl electrode (Fig. 1B). In a proof-of-concept study focusing on monitoring the biofilm formation and functionality, we cultivated Pseudomonas aeruginosa, a pathogenic bacterium secreting electroactive molecules known as phenazines when reaching high cell density. Subsequently, we monitored biofilm formation over 30 hours by repetitively measuring the electrochemical signal using differential pulse voltammetry (DPV). The analysis of anodic (Fig. 1C) and cathodic (Fig. 1D) currents unveiled an increasing oxidation peak at -0.28V vs Ag/AgCl, varying among the five electrodes, attributed to phenazine formation (Fig. 1E). Furthermore, both anodic and cathodic currents decreased at the negative applied potential (-0.8 V vs Ag/AgCl), correlating with bacterial growth and phenazines production (Fig. 1F). Electrochemical impedance spectroscopy (EIS) at different DC potentials (-400, +50, and +400 mV vs Ag/AgCl; Fig. 1G, +400mV) was performed, and the impedance generated was correlated with 10 different equivalent electrical circuits (optimal circuit with the best fitting score is shown in Fig. 1H) [2]. Hourly monitoring of biofilm growth was conducted using microscopy imaging at multiple locations along the microfluidic channel (Fig. 1I, image after 10hr). The experiment's conclusion involved validating biofilm formation through fluorescent staining of the biofilm's sugars (Figs. 1J – 1L, phase image, fluorescent image, and overlay image). By integrating the developed electrochemical micro-system with microscopy imaging, real-time and direct monitoring of biofilm formation becomes achievable. Furthermore, this integrated system can be employed across electrochemical and microscopic platforms to explore biofilm properties in response to diverse conditions.

Figure 1. (A) Microfluidic electrochemical array illustration (B) photograph of the experimental setup (C) 30-hours anodic and cathodic (D) voltammograms recorded in the presence of Pseudomonas aeruginosa (PA14). (E) transient oxidation peak current at -0.28V (F) the anodic and cathodic currents at -0.8V (G) transient impedance spectrographs recorded from PA14 (H) raw data and fitted electric circuit. Microscopic imaging of biofilm growth at (I) 10 hr, end of experiment (J) phase image, (K) biofilm polysaccharides fluorescent staining and (L) overlayed image.
[1] Estrada-Leypon, O. et. al, Bioelectrochemistry 2015, 105, 56–64.
[2] Ben-Yoav, H. et.al, Electrochim. Acta 2011, 56 (23), 7780–7786.

Figure 1