Thaddaeus A. Webster

Electrochemical detection of Pseudomonas aeruginosa in human fluid samples via pyocyanin.

The ability to quickly detect the presence of pathogenic bacteria in patient samples is of the outmost importance to expedient patient care. Here we report the direct, selective, and sensitive detection of the opportunistic pathogen Pseudomonas aeruginosa, spiked in human whole blood with sodium heparin, urine, sputum, and bronchial lavage samples using unmodified, disposable carbon electrode sensors that detect the presence of pyocyanin, a virulence factor that is unique to this species. Square wave voltammetry scans of biological fluids from healthy individuals spiked with P. aeruginosa showed a clear pyocyanin response within one day of culturing at 37°C. Scans of supernatants taken from cultures of P. aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermis, and Bacillus cereus taken over a span of three days in the potential range from -0.5 to 0 V vs. an Ag/AgCl reference showed no electrochemically detectable molecules with the exception of P. aeruginosa. The results indicate the potential to sensitively and selectively determine the presence of P. aeruginosa in human samples via the electrochemical detection of pyocyanin in less than 5 min, without any sample preparation or separation steps.

AMPEROMETRIC DETECTION OF PYOCYANIN IN NANOFLUIDIC CHANNELS

Microfabricated nanofluidic electrode assemblies (NEAs) with integrated palladium references were used to amperometrically monitor changes in pyocyanin concentration. Pyocyanin is an electroactive molecule that is produced by the opportunistic pathogen Pseudomonas aeruginosa and is directly linked to cellular processes that increase both robustness and virulence in this bacterium. This is the first time that pyocyanin has been measured in real time using microfabricated sensors. A linear response in faradaic current (R2 = 0.96) was observed over a biomedically relevant range of pyocyanin concentrations (0–100 μM) while continuously measuring the current for 2 h. Measurement of the current that results from the repeated oxidation and reduction of pyocyanin at two closely spaced electrodes inside the device nanochannel yielded a 1.07 μM limit of detection without electrical isolation of the electrochemical cell. Since a reference electrode is integrated inside the nanofluidic channel of these sensors, they can potentially be employed to detect pyocyanin and other redox-active molecules in wide range of medical and environmental settings where space is limited. NEAs were also used with an external Ag/AgCl reference electrode to determine the concentration of pyocyanin in trypticase soy broth samples. This type of analysis is completed in less than 2 min and the detection limit was determined to be 441 nM.

Improved monitoring of P. aeruginosa on agar plates

Described is the fabrication of a disposable electrochemical assay that is integrated with standard Kings A agar culture plates, for the selective and specific detection of Pseudomonas aeruginosa. Agar plates provide several advantages over liquid culture, including protecting the sensor from biofouling and faster identification in small sample volumes. Cultures of P. aeruginosa, starting from initial cell counts of 102 to 108 cells in 5 microliter volumes, were incubated at 23, 37, and 42 °C and monitored both visually and electrochemically. Square wave voltammetry scans confirmed the production of a redox species, pyocyanin, over time that was dependent on the initial load of cells. The pyocyanin easily diffuses through the agar to reach the electrode surface. Using this simple and cheap approach, positive identification of P. aeruginosa was achieved several hours faster via electrochemical detection compared to traditional visual analysis.

Monitoring Pseudomonas aeruginosa in culture plates using embedded electrochemical sensors

Described is the fabrication of a disposable electrochemical assay, embedded in solid growth media agar. By applying a unique set of potentials and measuring current change using electrochemical sensors, we were able to selectively detect the presence of Pseudomonas aeruginosa. Bacterial cultures of P. aeruginosa, Escherichia coli, and Staphylococcus aureus were grown on these plates for a period of 48 hours at room temperature. The maximum currents from square-wave voltammetric scans showed the production of redox active molecules by P. aeruginosa after 15 hours of growth. This simple disposable electrochemical approach for detecting P. aeruginosa could have implications for diagnosing the presence of P. aeruginosa infections in limited resource areas.