C503041 1000 modified bradford protein assay kit

A scanning electron microscope (SEM, ProX, Phenom, Netherlands) was used for observation of biofilms on the electrode. The biofilms in the BES were incubated under potentiostatic conditions for 4 days, and after a subsequent cyclic voltammetry (CV) scan, the biofilm samples were obtained by cutting off the electrodes. Electrode samples were first washed in a 0.1 M phosphate buffer solution (pH 7.0) for 5 min, followed by hardening in 2.5 % glutaraldehyde solution for 5 h. Next, the samples were dehydrated in an ethanol gradient (10%, 30%, 50%, 70%, 90%, 95%, and 100%) and t-BuOH. Finally, after freeze-drying, the samples were coated with evaporated platinum before being viewed in the SEM with an operating voltage of 15 kV. The protein in the cells on the electrodes was dissolved and extracted using 0.2 M NaOH (Qin et al., 2020 (link)), followed by quantification with Coomassie blue staining using a protein quantification kit (C503041-1000 Modified Bradford Protein Assay Kit; Sangon Biotech, Shanghai, China).

The morphologies of the biofilms on the electrodes were characterized using scanning electron microcopy (SEM; S-3000N, Hitachi, Japan) and fluorescence microscopy (Axio Scope A1, Carl Zeiss, Germany). The carbon cloth electrodes with biofilms were removed after incubation for 3 days, gently washed in water, and then treated with glutaraldehyde (2.5%), osmic acid, and ethanol. After freeze-drying and coating with evaporated platinum, the washed biofilm samples were imaged using SEM. The cells were then dyed with 4′,6-diamidino-2-phenylindole before fluorescence microscopy imaging.
The biofilm was then quantified by extracting the total biofilm protein with a boiling 0.2 M NaOH solution (Zhao et al., 2013 (link)). A carbon cloth sample (2 cm × 0.66 cm) was placed into a 2 mL sealed tube, together with 0.5 mL NaOH and some glass beads, and the tube was then subjected to a super high-speed vortex (6.5 m s⁻¹, 45 s) using a homogenizer (FastPrep-24, MP, United States). The mixture was then centrifuged at 8000 g for 3 min, and finally, the supernatant was collected for protein quantification. The supernatant was then quantified by Coomassie blue staining via a protein quantification kit (C503041-1000 Modified Bradford Protein Assay Kit, Sangon Biotech, China).