Development and Application of a Novel Organic Crystal for Reagent-Free, In-Situ Electrochemical Sensing of Bacterial Viability

Open Access
- Author:
- Bolotsky, Adam
- Graduate Program:
- Materials Science and Engineering
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- December 15, 2020
- Committee Members:
- Seyedehaida Ebrahimi, Thesis Advisor/Co-Advisor
Morteza Kayyalha, Committee Member
Larry Cheng, Committee Member
John C Mauro, Program Head/Chair - Keywords:
- Biosensor
Bacterial viability
Electrochemical sensor
Redox-active
Organic crystal
Rapid antibiotic susceptibility testing
Reagent-free
Metabolic activity - Abstract:
- Monitoring bacterial viability is important in clinical, commercial, and academic settings. One of the important areas for measuring bacterial viability is antibiotic susceptibility testing (AST), which is critical in determining bacterial resistance or susceptibility to a particular antibiotic. Simple-to-use, phenotype-based AST platforms can assist care-givers in timely prescription of the right antibiotic. Monitoring the change of bacterial viability by measuring electrochemical Faradaic current is a promising approach for rapid AST. However, the existing works require mixing redox-active reagents in the solution which can interfere with an antibiotic’s effect. In this thesis, we developed a facile electrodeposition process for creating redox-active crystalline layers (denoted as RZx) on various graphitic carbon electrodes, including pyrolytic graphite sheet (PGS), laser induced graphene (LIG) on polyimide sheets, and screen-printed carbon electrodes on paper, and utilized them as the sensing layer for reagent-free bacterial monitoring. Effects of the conductive substrate and composition of the deposition solution on elemental composition, crystallinity, and morphological properties of RZx were studied. Our studies show that phenylalanine plays a critical role in formation of stable RZx crystals. In addition, compared to common metals, graphene-based substrates lead to a much higher crystal surface coverage. The sensors enable detection of bacterial metabolic activity/respiration mainly due to the pH-sensitivity of RZx (~ 53 mV/pH) and also oxidation of excreted redox-active metabolites from cells. By monitoring the differential voltammetric signals, the sensors enable accurate prediction of the minimum inhibitory concentration (MIC) of two model antibiotics (ampicillin or kanamycin) for Escherichia coli (E. coli) K-12 in 60 minutes (p < 0.03). The sensors are stable after 60 days storage in ambient conditions and enable analysis of microbial viability in complex solutions, as demonstrated in spiked milk and human whole blood. We have also demonstrated a proof-of-principle integrated sensor and microfluidic system using RZx/LIG electrodes to monitor bacterial viability in real time.