PyoPower: a self-powered pyocyanin electrochemical biosensor for the detection of Pseudomonas aeruginosa infections

Introduction: Pseudomonas aeruginosa is an opportunistic pathogen prevalent in wounds of hospital patients. Current detection methods for P.aeruginosa take between 24-48 hours through use of swab samples and plate cultures. During this time, the infection worsens and forms a biofilm, making the infection increasingly difficult to eradicate. Therefore, a more rapid sensing system would improve the treatment of these infected wounds. We aim to utilize the redox properties of pyocyanin, a key biomarker of P.aeruginosa, to create an electrochemical biosensor powered by enzyme fuel cells. Based on our hypothesis that pyocyanin is a potential electron acceptor for the pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) enzyme while PQQGDH oxidizes glucose, we designed a self-powered pyocyanin electrochemical biosensor for the detection of P.aeruginosa infections with the combination of glucose/pyocyanin driven enzyme fuel cell. These experiments lay the groundwork for a more rapid and efficient biosensing enzyme fuel cell system for P.aeruginosa in wound care.

Materials and

Methods: We recombinantly prepared Acinetobacter calcoaceticus derived PQQGDH (Igarashi et al., 2004), by constructing an expression vector (pET30c) containing a c-terminal polyhistidine tag (HisTag). The expression vectors were then transformed into Escherichia coli (E. coli) BL21 DE3 strain for protein expression. Transformants were grown in Luria-Burtani (LB) Media and protein expression was induced with IPTG. The protein was harvested and purified via affinity chromatography. SDS PAGE was performed for size validation.

Bare gold disc electrodes were mechanically, chemically, and electrochemically cleaned. For enzyme immobilization, 3 μL of PQQGDH mixed with 22 μL Ketjen black (KB) was prepared for the anode while bilirubin oxidase (BOD) mixed with KB at the same volumes was prepared for the cathode. Clean gold disc electrodes were coated three times with the corresponding enzyme/KB mixture, followed by 1-hour glutaraldehyde deposition and 20-minute tris soak.

The electrodes with immobilized PQQGDH were placed in cells with 5 mM glucose and Ag/AgCl reference electrodes. CV was applied at -0.7 V to 0.5 V potential at a spin rate of 200RPM and a scan rate of 100 mV/s. Each reading was taken at increasing concentrations of pyocyanin from 0-100 μM. The same was done for increasing concentrations of glucose from 0-20 mM with a constant pyocyanin concentration of 100 μM. Finally, the PQQGDH anodes were placed in a biofuel cell with BOD cathodes, and open circuit potentiometry (OCP) was used to measure the potential difference between the two electrodes. Multiple readings were taken with increasing concentrations of pyocyanin from 0-100 μM.

Results, Conclusions, and Discussions: PQQGDH was successfully produced and purified as a soluble protein. Pyocyanin was confirmed as an electron acceptor for PQQGDH via CV and OCP, as the PQQGDH electrodes demonstrated increasing current and potential with increasing concentrations of pyocyanin and glucose. Future experiments will further examine the relationship between power generation and glucose and pyocyanin concentrations as well as other factors like resistance, mixing rate (RPM), and scan rate (mV/s).

Acknowledgements (Optional): Molecular Engineering of PQQGDH and Its Applications”, Archives of Biochemistry and Biophysics, Satoshi Igarashi, Junko Okuda, Kazunori Ikebukuro and Koji SODE, 428(1), 52-63. (2004),