Fast Selective Detection of Pyocyanin Using Cyclic Voltammetry

Abstract

Pyocyanin is a virulence factor uniquely produced by the pathogen Pseudomonas aeruginosa. The fast and selective detection of pyocyanin in clinical samples can reveal important information about the presence of this microorganism in patients. Electrochemical sensing of the redox-active pyocyanin is a route to directly quantify pyocyanin in real time and in situ in hospitals and clinics. The selective quantification of pyocyanin is, however, limited by other redox-active compounds existing in human fluids and by other metabolites produced by pathogenic bacteria. Here we present a direct selective method to detect pyocyanin in a complex electroactive environment using commercially available electrodes. It is shown that cyclic voltammetry measurements between -1.0 V to 1.0 V reveal a potential detection window of pyocyanin of 0.58-0.82 V that is unaffected by other redox-active interferents. The linear quantification of pyocyanin has an R² value of 0.991 across the clinically relevant concentration range of 2-100 µM. The proposed method was tested on human saliva showing a standard deviation of 2.5% ± 1% (n = 5) from the known added pyocyanin concentration to the samples. This inexpensive procedure is suggested for clinical use in monitoring the presence and state of P. aeruginosa infection in patients.

Keywords: cyclic voltammetry; diagnosis; electrochemical detection; pyocyanin; quorum sensing.

Figure 1

Cyclic voltammogram of 500 µM pyocyanin (blue) compared to the MilliQ water control (black) using a scan rate of 0.05 V/s. No signals are generated from cyclic voltammetry of MilliQ water, while pyocyanin reveals a characteristic profile with oxidation peaks at −0.560 V, −0.311 V and 0.699 V (arrows) measured versus the Ag reference electrode.

Figure 2

Potential peak values of the different compounds and related detection width (bars) extracted from cyclic voltammograms of the individual compounds. The detection window of pyocyanin with peak at 0.699 V and corresponding start and end potentials lies outside the interaction width of the other redox-active compounds.

Figure 3

Cyclic voltammograms of mixture of compounds containing pyocyanin (mix-1, red curve) and mixture without pyocyanin (mix-2, black curve) measured versus the reference electrode. The red curve has a peak at 0.68 V confirming the presence of pyocyanin (arrow), while it is absent in the black curve of mix-2.

Figure 4

Detection of pyocyanin in different mix backgrounds between 5 µM and 100 µM in addition to pyocyanin detection in MilliQ water (zero mix background). The current peaks of the varying pyocyanin concentrations were unaffected by the mix concentration. Detection of 2 µM pyocyanin was only possible when no interfering signals were present. When the mix concentration was above 25 µM the detection limit of pyocyanin remained 6 µM.

Figure 5

Detection limit of pyocyanin using max peak currents of cyclic voltammograms. The limit of detection (LOD) is 2 µM. The linear fit to peak currents vs. pyocyanin concentration has a linearity of 0.991. The error bars represent the standard deviation from measurements in the four different backgrounds of mix concentrations and in no mix background.

Figure 6

Peak potential as function of pyocyanin concentration. The potential peaks go towards the lower detection window of pyocyanin as the pyocyanin concentration decreases.

Figure 7

Example of pyocyanin detection in human saliva sample. Black illustrates a cyclic voltammogram of human saliva without pyocyanin. Dark red cyclic voltammogram is of human saliva containing pyocyanin and mix. The peak around 0.60 V versus the reference electrode confirms the presence of pyocyanin in the sample (arrow).