Spectroelectrochemical monitoring of resazurin was performed with SPELEC, a fully integrated instrument for spectroelectrochemistry that combines both the electrochemical (bipotentionstat/galvanostat) and spectroscopic components (UV-Vis light source and detector) in a portable system. Even though SPELEC contains a light source with a wavelength range of 200 nm to 900 nm, a 395 nm LED (LEDVIS395) was used as the excitation light source. This LED has a well-defined range of light emission, avoiding any contribution of light in wavelengths where Alamar Blue fluoresces.

The rest of the setup consisted of a reflection probe (RPROBE-VIS-UV), a spectroelectrochemical reflection cell for Thin-Layer Flow-Cell Screen-printed electrodes (TLFCL-REFLECELL) and the components included in the fluorescence kit (FLKIT). The FLKIT contains two short optical fibers (600 μm), two optical filters (one of which filters wavelengths between 230–500 nm, and the other filters wavelengths between 300–750 nm), and two holders for the filters. The total setup is shown in Figure 1.

The Thin-Layer Flow-Cell Integrated Screen-Printed Circular Carbon Electrode (TLFCL110-CIR) consists of a flat ceramic card containing the electrochemical cell with three-electrode system (4 mm diameter circular carbon working electrode, a carbon counter electrode, and a silver pseudo-reference electrode, Figure 2). This electrodic platform is covered by a slide with a channel that limits the thickness of the solution to 400 μm.

SPELEC was controlled with DropView SPELEC, a dedicated software that provides spectroelectrochemical information and includes tools to perform adequate treatment and analysis of the collected data. All hardware and software used for this study is compiled in Table 1.

In order to reduce the resorufin (generated in the previous step) to dihydroresorufin, the next potential pulse was performed by applying -1.00 V for 300 s. Figure 5a displays the electrochemical signal (grey line) as well as the evolution of the fluorescence band at 590 nm with time (green line). Figure 5b shows the fluorescence spectra recorded during this second step.

Although the intensity of the emission band at 590 nm was completely stable at the end of the first step (Figure 4a, green line), the second step demonstrates that this emission band decreases abruptly for 100 s when a potential of -1.00 V is applied. There was no fluorescence detected during the rest of the experiment, demonstrating the successful reduction of resorufin (highly fluorescent) to dihydroresorufin, a non-fluorescent molecule (Figure 3).

Finally, a third potential step was applied with the goal of reoxidizing the dihydroresorufin generated in the second step back to resorufin. Figure 6a shows the electrochemical signal (grey line) and the spectra evolution of the fluorescence band centered at 590 nm (green line) when -0.10 V was applied for 300 s. This graphic shows that the optical signal reaches the fluorescence values obtained in the first pulse of the chronoamperometry experiment, shown in Figure 4a.

The spectra evolution displayed in Figure 6b confirms the increase of the fluorescence band centered at 590 nm. Dihydroresorufin is completely oxidized back to resorufin because the fluorescence maximum observed in the first step of the experiment (Figure 4b) is also obtained here in the third step. Therefore, the reversibility of the redox process between resorufin and dihydroresorufin is clearly demonstrated.