A voltammetric flavin microelectrode for use in biofilms

Abstract

Biofilms used in bioelectrochemical systems are expected to transfer electrons using electron transfer mediators. One mediator type, flavins, which includes flavin mononucleotide, riboflavin, and flavin adenine dinucleotide, has been found to be endogenously produced by Shewanella oneidensis MR-1. However, the presence and concentration of flavins inside a S. oneidensis MR-1 biofilm have never been reported. The goal of this study was to develop a flavin microelectrode capable of measuring flavins inside a living biofilm and apply it to a biofilm which produces flavins. Because flavins are electrochemically active molecules, the flavin microelectrode was based on detection via square-wave voltammetry. The microelectrode consisted of a carbon working electrode with a 10–30 μm tip diameter, a built-in platinum counter electrode, and a Ag/AgCl reference electrode, all enclosed in a glass outer case. The microelectrode was calibrated between 0.1 μM and 10 μM flavins and showed a linear correlation between flavin concentration and peak currents located at −424 mVAg/AgCl on a square-wave voltammogram. We also developed a model to explain the electrochemical mechanism of flavin detection, and to determine the effective surface area of the microelectrode, the standard reduction potential, and the transfer coefficient. We found that the effective surface area of the microelectrode was close to 100 times the projected surface area. The model predicted a standard reduction potential for RF/RFH2 of −419 mVAg/AgCl at 20 °C and a transfer coefficient of 0.45. Lastly, we measured flavin concentration inside a S. oneidensis MR-1 biofilm grown on a glass surface using oxygen as the electron acceptor. The flavin concentration reached 0.7 μM, increasing near the bottom of the biofilm, where no oxygen was present. This shows the possibility that flavins are produced in the anaerobic zone to act as intermediate electron acceptors in the deeper parts of the biofilm, where there is no oxygen.

Electrochemically active biofilms: facts and fiction. A review

“…This is because when the potential pulse straddles the standard reduction potential, the forward pulse produces an anodic current and the reverse pulse produces a cathodic current. The ratio of the peak forward current to the peak reverse current is 0.91, reflecting the quasi-reversible nature of the reaction (Nguyen et al 2012b). The combination of the forward, reverse, and difference currents in SWV makes it a useful tool in analyzing electrochemically active species.…”

“…For example, microelectrodes have been used to measure concentrations of oxygen, hydrogen, hydrogen sulfide, and carbon dioxide, as well as pH, redox potential, and local flow velocities (Lee and Debeer 1995; Yang and Lewandowski 1995; Xia et al 1998; Yu and Bishop 1998; Beyenal et al 2004). Voltammetric microelectrodes that use techniques such as cyclic voltammetry and pulse voltammetry have also been used to detect chemicals such as metal cations and flavins (Xu et al 1998; Nguyen et al 2012b). Because microelectrodes are minimally invasive and have dimensions that can be as small as 1-5 mm, they are well suited to studying changes in both the EAB and the bulk solution above the EAB during electrode respiration.…”

Modeling biofilms with dual extracellular electron transfer mechanisms

“…These are identical to the parameters used in our previous study, which used squarewave voltammetry to detect flavin concentrations inside S. oneidensis biofilms. 53 Figure 10 shows the simulated squarewave voltammetry results. The shapes of the differential-current voltammograms are nearly identical regardless of the EET mechanisms.…”

“…To avoid jump discontinuities when squarewave simulations were run, which would prevent model convergence, the voltammetric signal changes between peak and trough potentials were approximated by a differentiable, and thus continuous, spline function consisting of two quarter-sine waves connected by a straight line, as developed in Nguyen et al . 53 and shown in Figure 2. This was chosen over a Fourier series expansion to avoid Gibbs ringing artifacts in the model.…”

Microbiosensor for the detection of acetate in electrode-respiring biofilms

“…The carbon wire used to construct microbiosensors was a 30-μm-diameter electrochemically activated carbon fiber (World Precision Instruments, Sarasota, FL, USA, catalog #C3005). The construction of the glass capillary – sealing the carbon wire, covering the carbon wire with glass, making the glass outer case out of a pulled Pasteur pipette, and making the silver/silver chloride reference electrode – has been detailed in the SI and previously (Nguyen et al, 2012). Following the construction of the microbiosensor parts, the microelectrode tip (diameter: 30 μm) and the glass outer case tip (diameter: 30 μm) were first assembled under 40 × microscope magnification using micromanipulators.…”