(101g) Monitoring Pyocyanin Production by Bacteria Using Nanofluidic Electrochemical Sensors

While processes for manipulating and exploiting bacteria are well known, and have been used for hundreds of years, studies of the interactions between microbial organisms have only begun relatively recently [1]. Of particular interest is the detection of quorum sensing molecules excreted by bacterium that coordinate both inter- and intraspecies interactions. The main objective of this project is to monitor the secretion of the quorum sensing molecule pyocyanin by P. aeruginosa cells as they are exposed to various stressors.

It is known that pyocyanin is an electrochemically active molecule involved in regulating cellular response in its own species, other microbes, and even mammalian cells [2]. We therefore plan to employ nanofluidic electrochemical detection (NED) sensors to detect the local concentration of pyocyanin surrounding microbes while optically monitoring the cellular response. These solid-state devices consist of a 50 nm high solution-filled cavity bounded by two parallel electrodes in which electrochemically active molecules transfer charge between suitably biased electrodes. Electrochemical reactions typically only involve one or a few electrons per molecule rendering direct detection at low concentrations virtually impossible. By utilizing electrochemically active molecules, which can repeatedly undergo reversible reduction and oxidation, each molecule can transfer, on average, thousands of electrons between the electrodes while residing in the cavity [3]. The resulting current is proportional to the concentration of the target molecule.

As a proof of concept, we first tested the ability to detect pyocyanin using commercial redox cycling devices in Phosphate Buffer (PB) and Lysogeny Broth (LB) growth media. Interdigitated electrode array sensors were used to detect pyocyanin concentration in PB and LB in the range of 0.1 – 40 µM. The concentrations detected are physiologically relevant and indicate that sensors based on redox cycling can be used in microbial experiments. Future work will employ NED sensors integrated in microfluidic channels to measure pyocyanin production by P. aeruginosa under various conditions.

References:

[1] Shiner, E.K., et al. (2005) 29, 935-947

[2] Allen, L., et al. J Immunology (2005) 174, 3643; Bukelman, O., et al. Chem Commun (2009) 2836.

[3] Goluch, E.D., et al. Anal Bioanal Chem (2009) 394, 447; Goluch, E.D., et al. The 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences (microTAS) (2010) 482.