Development of electrochemical DNA-based biosensors for the detection of Shiga toxin-producing E. coli (STEC)
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Date
2022
Authors
Wasiewska, Luiza Adela
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University College Cork
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Abstract
Shiga toxin-producing E. coli (STEC) is a food-borne pathogen of significant public health concern, due to the severity of the illness it can cause including severe bloody diarrhoea and haemorrhagic uremic syndrome. A key pathogenicity factor is the ability to produce Shiga T Toxin 1 and 2, which are encoded by genes stx1 or stx2. These genes are key targets in molecular-based assays to detect this group of pathogens. However, many of these assays, including real-time PCR approaches are considerably time-consuming and there is a need for a more rapid screening assay which could be used in agri-food settings. This publication-based thesis presents the development of DNA based electrochemical sensor as an alternative approach for the rapid detection of the stx1 or stx2 genes and explores the application of gold interdigitated micro electrodes (IDEs) for electrochemical pH control and redox molecule accumulation.
Firstly, a comprehensive literature review was undertaken regarding the current STEC detection approaches, challenges presented, and the opportunities for the development of electrochemical sensors to detect this group of pathogens. A detailed analysis of gene sequences used for targeting both general E. coli and STEC was performed and the recently developed electrochemical nucleic acid-based sensors were classified based on the electrode’s material used and its modification. This literature review allowed the selection of the most promising approach for the development of the DNA sensor in this thesis.
Initial work focused on using reporter DNA tagged with silver nanoparticles that could subsequently be oxidised. The aim being that silver ions detected electrochemically could then be correlated to the DNA present in a sample. This began with the development of an electrochemical sensor for silver ions detection using
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electrochemical pH control. In this approach, one interdigitated electrode (IDE) comb was used as a working electrode, while the other was used as a generator electrode that produced protons, subsequently decreasing the local pH. The combination of silver ions complexation with chloride and in-situ pH control resulted in a linear calibration range between 0.25 and 2 μM in tap water and a calculated limit of detection (LOD) of 106 nM without the need to add either acid or supporting electrolytes. However, even though the LOD of the silver ions detection sensor was satisfactory for their detection in tap water, it was not sufficiently sensitive for use with the DNA sensor. Therefore, the approach for DNA detection was changed, and the focus was moved to the use of methylene blue instead of silver nanoparticles.
In this work, a highly sensitive, label-free, electrochemical DNA-based sensor for the detection of the stx1 gene was developed. Firstly, a working IDE was modified with gold nanoparticles and chitosan-gold nanocomposite allowing immobilisation of amine-modified probe DNA. Label-free electrochemical detection was undertaken using methylene blue as a redox molecule, which intercalated into the double-strand DNA. An accumulator IDE was used for the accumulation of methylene blue around the sensor IDE by applying an open circuit potential during the incubation. Reduction of methylene blue was recorded using square wave voltammetry. Using this label-free detection, a linear response was shown at concentrations ranging from 10-6 M to 10-16 for synthetic stx1 target strands, with the lowest LOD of 10-16 M. Chromosomal DNA extracted from four different STEC E. coli strains was used to confirm the selectivity of the presented method.
Finally, a multiplex sensor for the simultaneous detection of two genes coding for toxin production, stx1 and stx2, was developed. The LOD was further improved by three orders of magnitude, upon deposition of a thicker layer of gold nanoparticles and
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re-optimisation of chitosan-gold nanocomposite deposition. The probes complementary to stx1 and stx2 were immobilised on the same chip allowing for multiplex detection. The modification of the surface has allowed for decreasing the LOD for both target genes to 10-19 M instead of 10-16 M. The multiplex sensor was validated by the detection of chromosomal DNA extracted from bacterial culture as well. Such a multiplex sensor, if combined with on-chip DNA extraction, could revolutionise the point-of-use detection of STEC as well as other pathogens for instance on-farm or in the food industry.
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Keywords
Shiga toxin-producing E. coli , Biosensor , DNA-based detection , Interdigitated electrodes , Methylene blue , Electrochemistry , Electrochemical pH control
Citation
Wasiewska, L. A. 2022. Development of electrochemical DNA-based biosensors for the detection of Shiga toxin-producing E. coli (STEC). PhD Thesis, University College Cork.