Electrochemical Detection of Viable Bacterial Cells Using a Tetrazolium Salt

Abstract

In this study, electrochemical detection of viable bacterial cells was performed using a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which was converted to an insoluble and redox active formazan compound in viable microbial cells. The insolubility of this formazan was effectively exploited as a surface-confined redox event. An indium-tin-oxide electrode was applied to a microbial suspension that had been incubated with MTT and was heated to dry for the extraction and adsorption of formazan. Drying led to the appearance of a distinctive voltammetric oxidation peak at +0.1 V vs Ag|AgCl, the magnitude of which was successfully correlated to the number of viable microbes in the suspension. Thus, the electrochemical detection of formazan was effectively coupled with the thermal lysis of microbes. It is also noteworthy that this lysis-adsorption technique was highly selective to the hydrophobic formazan molecule due to the removal of hydrophilic cell components during equilibration in a phosphate buffer before voltammetric measurement. This technique was capable of detecting microbes above 2.8 × 10 CFU mL and required only a 1 h incubation. The results of this study indicate that the sensitivity of the present technique is up to 10 000-fold higher than that of MTT colorimetry. The higher sensitivity was mainly ascribed to the concentration of the microbially produced formazan on the electrode by thorough desiccation of the bacterial suspension.

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“…Bacterial detection techniques usually employ antibodies (labelled or un‐labelled), enzymes, DNAs or even a whole cell (micro‐organism) as the bioreceptors, but also redox mediator‐assisted electrochemical sensing has been reported . The classical procedure of living bacterial cell detection with redox indicators is based on the metabolic consumption (cellular reduction) of the redox indicator (i. e., a compound that can be reduced by the uptake of an electron) by the respiratory electron transport chain of living bacterial cells . The consumption of the redox indicator is time‐dependent and can be used for the detection and quantification of living bacterial cells in a liquid sample .…”

“…[76] The classical procedure of living bacterial cell detection with redox indicators is based on the metabolic consumption (cellular reduction) of the redox indicator (i. e., a compound that can be reduced by the uptake of an electron) by the respiratory electron transport chain of living bacterial cells. [77,78] The consumption of the redox indicator is time-dependent and can be used for the detection and quantification of living bacterial cells in a liquid sample. [44] This concept has been known and is well-adopted in colorimetric bacteria assays, but recently also found application in electrochemical sensing as many of the known metabolic activity indicators are electro-active.…”

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“…[71] The insolubility of formazan was credited for the high sensitivity obtained in this study, because a high concentration of the formazan could be concentrated through desiccation and adsorption as well as immobilized on the surface without the risk of diffusion in solution during voltammetric readings, thereby leading to a strong adsorption intensity peak. [71] Besides the detection of MRSA, V. Parahaemolyticus, and E. Coli, the non-resistant strains of S. Aureus also contributes to a wide spectrum of diseases, ranging from skin infections to pneumonia, meningitis and sepsis, hence reaffirming the need for its early detection and therapy. [72] Compared to other bacterial species, the detection of S. Aureus is more tricky owing to the thick polysaccharide layer and the limited number of externally exposed surface antigens.…”

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“…This method uses the tetrazolium salt 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) as a redox indicator for pathogen detection (Figure 7c,d). [ 170 ] The bacterial culture was incubated with MTT for 1 h to allow for intracellular uptake of the dye. Once intracellularly, the dye was converted to insoluble and redox‐active formazan.…”