Enhanced Electrochemical Impedance Spectroscopy Analysis of Microbial Biofilms on an Electrochemically In Situ Generated Graphene Interface

Biofilms can cause many bacterial diseases, such as dental disease. An in vitro detection of biofilms may help to screen antibiofilm drugs. An impedance measurement based on an Au electrode has been successfully used for in vitro real-time monitoring of animal and human cell growth. However, microbial growth on the Au electrode produced a poor signal because of the small size of microbial cells. We have recently demonstrated that graphene derivatives can be produced on a carbon electrode through facile electrochemical activation, thus forming a reduced graphene oxide-carbon electrode (rGO-CE). Based on this fact, we hypothesized that an in vitro formed rugose graphene layer of rGO-CE may provide a large surface area for the growth of microbial biofilms and can therefore produce a strong impedance signal in response to a change in the biomass. In this study, three oral bacteria, Streptococcus mutans ( S. mutans ), Actinomyces viscosus ( A. viscosus ), and Lactobacillus fermentum ( L. fermentum ), were cultured on the surfaces of rGO-CE. As a result, the impedance response signal of the rGO-CE for the growth of S. mutans and A. viscosus was found to be 3.3 times and 6.0 times stronger than that of the Au electrode at 1.17 and 54.7 kHz, respectively. In particular, the poorly adhering strain of L. fermentum also produced a detectable signal on the graphene electrode but not on the Au electrode at 1.17 kHz. Furthermore, destructions of the biofilms grown on the rGO-CE by cetylpyridinium chloride were successfully monitored by impedance changes. Overall, it is promising to develop a graphene-based impedance biosensor platform for biofilm study and antibiofilm drug screening.