Three-Mediator Enhanced Collisions on an Ultramicroelectrode for Selective Identification of Single Saccharomyces cerevisiae

Selective detection of colliding entities, especially cells and microbes, is of great challenge in single-entity electrochemistry. Herein, based on the different cellular electron transport pathways between microbes and mediators, we report a three-mediator system [K₃Fe(CN)₆, K₄Fe(CN)₆, and menadione] to achieve redox activity analysis and selective identification of single Saccharomyces cerevisiae without the usage of antibodies. K₄Fe(CN)₆ in the three-mediator system will oxidize near the electrode surface and increase the local concentration of K₃Fe(CN)₆, which will promote the redox reaction of S. cerevisiae. The hydrophobic mediator─menadione─can selectively penetrate through the S. cerevisiae membrane and get access to its intracellular redox center and can further react with K₃Fe(CN)₆ in the bulk solution. In contrast, the mediator can only get access to the bacterial membranes of Escherichia coli and Staphylococcus aureus, which results in different electrochemical collision signals between the above microbes. In the three-mediator system, upward step-like collision signals were observed in S. cerevisiae suspension, which are related to their microbial redox activity. In comparison, E. coli or S. aureus only generated downward current steps because the blockage effect of mediator diffusion suppresses their redox activities. When S. cerevisiae co-existed with E. coli or S. aureus, transients generated by both blockage and redox activity were observed. The approach enables us to trace the collision behaviors of different microbes and distinguish their simultaneous collisions, which is the foundation for further application of electrochemical collision technique in the specific identification of single biological entities.