Integrated Biosensor Platform for Detection of Waterborne Pathogens: Improving Public Health

Abstract

Waterborne diseases are caused by microorganisms known as pathogens e.g., bacteria, viruses, protists etc. that are commonly spread through contaminated fresh water sources. Diseases caused by these pathogens are today one of the leading causes of infection and mortality. In low- and middle-income countries public health is steadily becoming an increasing risk, with increased development directly producing waste pollution carrying pathogens into freshwater sources. Examples of freshwater sources include reservoirs, rivers and wells with waste pollution being due to poor sanitation, agricultural runoff, illegal dumping etc. The result of this pollution is being further exacerbated by poor wastewater infrastructure, reduced public understanding as well as lack of diagnostic and monitoring systems. One strategy of improving public health is to develop small portable devices that are easy-to-use to monitor levels of waterborne pathogens in drinking water to avoid unnecessary disease from contaminated sources. To do this we aimed to develop an integrated biosensing device for the sensitive detection of highly selective DNA sequences related to several waterborne pathogens.
The first study focusses on the development and optimisation of a biosensor assay using electrochemical impedance spectroscopy (EIS). The work looks at the optimisation of surface chemistry taking advantage of the passive adsorption of thiol groups to form a self-assembled monolayer (SAM). This is not only a relatively simple way of forming a sensing layer, but with the use of re-usable gold electrodes and E. coli specific thiolated ssDNA probes it is also cost-effective to optimise. This study also highlights the importance of different cleaning techniques for reusable gold electrodes and how this can impact ideal SAM formation for increasing probe-target interaction. The results of this study will act as a baseline for the ability of thiol self-assembled DNA probes to detect target sequences without any additional signal amplifying steps.
The second study takes the assay from the first study a step further by integrating a post-signal amplification step using a redox-active intercalator. Intercalators are molecules that bind between the base pairs of the DNA duplex structure. In doing so, they can alter the secondary structure of the duplex and increase the electrostatic field. This particular work exploits a novel redox-active intercalator called cobalt-aqphen, [Co(GA)2(aqphen)]Cl. The cobalt acts as the redox-active ligand of the compound, while the addition of the extended planar aqphen ligand with conjugated anthraquinone which has been shown to increase binding affinity between the base pairs. Intercalation of this molecule enables the potential for amperometric and non-faradaic detection of target DNA sequences at much lower limits of detection (LOD) without the need for an additional redox couple.
The third study develops a highly sensitive and selective assay integrating CRISPR/Cas-based SHERLOCK detection integrated with isothermal amplification, a conductive-polymer surface chemistry, peptide nucleic acid (PNA) probes and amperometric detection of TMB precipitation for signal-off function detection. This assay was used to demonstrate detection of both synthetic E. coli sequences as well as single-molecule SARS-CoV-2 viral RNA from unprocessed patient saliva. This assay can also be easily modified by altering the guide RNA that programmes the CRISPR/Cas enzyme to detect any target pathogen RNA. This provides great potential for multiplexed detection of multiple pathogen markers of clinically and environmentally relevant pathogens from a single point source sample.
In conclusion, this thesis aims to describe methods of increasing the sensitivity and selectivity of target nucleic acid detection using methods of pre-signal amplification such as isothermal amplification integrated with CRISPR/Cas SHERLOCK diagnostics as well as post-signal amplification using redox-active intercalator molecules. It also aims to explore ways of combining these techniques together to create truly integrated, cost-effective ways of detecting multiple waterborne pathogens of interest.

Date of Award	29 Mar 2023
Original language	English
Awarding Institution	University of Bath
Sponsors	Natural Environment Research Council
Supervisor	Pedro Estrela (Supervisor) & Mirella Di Lorenzo (Supervisor)