A viability assay for Candida albicans based on the electron transfer mediator 2,6-dichlorophenolindophenol

Abstract

Candida albicans is an opportunistic fungal pathogen with comparably high respiratory activity. Thus, we established a viability test based on 2,6-dichlorophenolindophenol (DCIP), a membrane-permeable electron transfer agent. NADH dehydrogenases catalyze the reduction of DCIP by NADH, and the enzymatic activity can be determined either electrochemically via oxidation reactions of DCIP or photometrically. Among the specific respiratory chain inhibitors, only the complex I inhibitor rotenone decreased the DCIP signal from C. albicans, leaving residual activity of approximately 30%. Thus, the DCIP-reducing activity of C. albicans was largely dependent on complex I activity. C. albicans is closely related to the complex I-negative yeast Saccharomyces cerevisiae, which had previously been used in DCIP viability assays. Via comparative studies, in which we included the pathogenic complex I-negative yeast Candida glabrata, we could define assay conditions that allow a distinction of complex I-negative and -positive organisms. Basal levels of DCIP turnover by S.cerevisiae and C. glabrata were only 30% of those obtained from C. albicans but could be increased to the C. albicans level by adding glucose. No significant increases were observed with galactose. DCIP reduction rates from C. albicans were not further increased by any carbon source.

Direct electrochemical determination of Candida albicans activity

“…In our previous study, we have developed a mediated bioelectrochemical system for the detection of C. albicans's viability in which electrons are shuttled from the NADHdehydrogenases of ETC (Hassan, 2011). Recently the early detection of C. albicans biofilms at porous electrodes has been reported (Congdon, 2013).…”

“…10 μl of the WST reagent was added to each well of a 96-well microtiter containing 170 μl of yeast cell suspension in YPD culture medium. After 20 min incubation at room temperature, the absorbance was measured at 450 nm using plate reader (Hassan and Bilitewski 2011).…”

Bioelectrochemical Systems for Measuring Microbial Cellular Functions

“…For the electrochemically‐inactive organisms, the direct communication with the electrode is not possible. Therefore, exogenous agents (artificial redox mediators) are required to wire the microbe‐electrode interactions through what is known as mediated electron transfer (MET) system .…”

“…Thus, as shown in Figure , the MET can be obtained using exogenous redox compounds (artificial electron shuttles) . MET is the most common BES, and its mechanism is clear, therefore, its use for the detection of microbes has been widely applied .…”

A Highly Selective Biosensor with Nanomolar Sensitivity Based on Cytokinin Dehydrogenase

“…2,6-dichlorophenolindophenol (DCPIP) is widely known as a photometric pH and redox indicator, and its membrane permeability makes it a popular redox coupling agent in assays of bacteria [21]–[23] and bacterial biofuel cells [24]. DCPIP modified electrodes have also been applied for amperometric determination of NADH [25]–[28] and ascorbic acid [29].…”