Bacterial sensors and controllers based on organic bioelectronics

Abstract

Bacterial infections and contaminations are worldwide problems, leading to morbidity and mortality, food waste and economic losses in a variety of industries. The situation is aggravated by the increased occurrence of antibiotic-resistant strains, identified by the WHO as one of the biggest threats to development, food security and public health today. The solution to this problem is complex and requires efforts from several different layers of the society, and different disciplines. The knowledge about microbiology has greatly advanced in the last decades and several powerful methods were introduced. However, in most clinical microbiology laboratories, culture-based techniques are still standard practice, representing a bottleneck in the diagnostic workflow. In this thesis, we prototype novel methods to detect and identify bacteria, aiming to reduce the time and workload for future microbiology research and diagnostics. Furthermore, a new methodology is devised to evaluate antimicrobial surface properties for relevant high-touch surfaces.

In Paper I, we investigated whether conducting polymers can be applied for label-free electrochemical detection of bacteria. Employing a poly(3,4-ethylenedioxythiophene): polystyrenesulfonate (PEDOT:PSS)-based two-electrode sensor we demonstrated that potentiometric detection and quantification of Salmonella Enteritidis is possible within 15 min, without any sample pre-treatment. We show that the reduction of PEDOT:PSS electrode occurs by low molecular weight species secreted by Salmonella Enteritidis. To evaluate the genericity of the sensor, several uropathogenic strains were tested and we found that they could all be detected using the sensor. In its current form, the sensor is a prototype, and we aim to improve its sensitivity and introduce specificity. Electroactivity was shown to be a rather common characteristic of bacteria and consequentially, electrochemical methods for detection and characterization of microbes are gaining momentum. We envision that this field will provide novel diagnostic devices but also contribute to discoveries in basic science.

Luminescent conjugated oligothiophenes, called optotracers, have previously been applied in microbiology to visualize extracellular matrix components in biofilms of Salmonella and Escherichia coli. In Paper II, we investigated the use of optotracers for detection and visualization of Staphylococcus aureus (S. aureus). We show that the optotracer HS-167 selectively binds to Staphylococci and can be used for fluorometric detection and quantification of S. aureus, as well as for staining and visualization using confocal microscopy. HS-167 displays an on-switch of fluorescence upon binding and it does not affect bacterial growth, which enabled us to develop a high-throughput assay where the fluorescence was plotted against bacterial density, measured as an increase in turbidity. The resulting slope was a quantifiable variable that we employed to compare binding of HS-167 to different species and strains. Diverse approaches collectively pointed to the cell envelope as the target for HS-167 binding. Finally, we showed that binding is highly dependent on the environmental conditions and those can be adjusted to tune the selectivity of HS-167.

To improve optotracer design for detection of S. aureus, a better insight into the structure- function relationship is needed. In Paper III, we set out to establish a structured approach to optotracer screening that would enable us to compare optotracer performance. As we compared a library of ten different optotracers, we identified the length to be positively correlated and the total negative charge to be negatively correlated with the ability to detect S. aureus. A balance between the two was necessary to achieve the highest signal while maintaining selectivity. Selected optotracers were added to S. aureus and visualized under the confocal microscope. All localized in the cell envelope of the bacterium, as was previously observed for HS-167 (Paper II). We foresee that further insight into the binding mechanism will enable targeted optotracer design, and together with optimized assay conditions, specific detection of different bacterial species.

Copper is known to possess antimicrobial properties, yet studies have reported discrepant results on its efficacy, especially in the clinical settings. Disagreeing results were ascribed to the lack of a standardized approach to evaluate the antimicrobial properties of copper surfaces. In Paper IV, we establish a multifaceted approach to address the effect of human touch, which we simulate by applying artificial sweat, on surface corrosion and antimicrobial properties of copper. We found that artificial sweat accelerates corrosion, leading to changes in surface appearance and wettability. Corrosion did not negatively affect the antimicrobial properties of copper as these surfaces killed bacteria within minutes, regardless of ageing or corrosion product formation. The antimicrobial effect is ascribed in part to copper ions released from the surface and in part to direct surface contact. To further validate the results of this study, other bacterial species need to be tested. Since high touch surfaces are likely to collect a lot of microbes over time, it would be of interest to determine how the bacterial load affects the antimicrobial properties.