Electrochemical biosensor development for enhanced detection of pathogens which cause sepsis and hyperinflammation

Sepsis represents a significant and growing challenge within modern healthcare settings. It can deteriorate quickly, potentially leading to fatality. The common characteristic to all sepsis patients is the importance of pathogen identification. Currently, pathogen detection is lengthy (hours to days) and as such is detrimental to patient outcome. Rapid pathogen detection will aid clinicians in source control and help guide targeted treatment in shorter time scales giving patients the best chances for recovery. Electrochemical biosensors have potential to offer a route to rapid, low cost, multiplexed pathogen identification. The work presented in this thesis represents a series of investigations made to apply electrochemical methods to rapid pathogen detection at the point of care. The first section presents a literature review exploring sepsis, current detection methods employed and identifying the requirements of an electrochemical biosensor for pathogen detection. The second section details investigations into suitable platforms on which to construct electrochemical biosensors. Various electrode platforms were investigated and the results from these experiments were used to design custom ‘printed circuit board’ electrodes. The ability to form sensing layers composed of biorecognition elements on printed circuit board electrode surfaces is then demonstrated using Streptococcus pneumoniae genomic DNA to form a S. pneumoniae recognition surface. lytA was detectable on this S. pneumoniae DNA biosensor in serum samples at 1 pM after 15 minutes of sample addition at room temperature. Finally, the development of a simple enzyme-based sensor for SARS-CoV-2 is demonstrated. Detection performances are described for multiple positive and negative controls culminating in clinical sample testing. Viral PCR sample solution clinical samples are shown to be statistically different from a negative sample (P = 1.2E-7). Viral transport medium clinical samples showed multiple positive and negative samples were significantly different with preliminary calculations showing a sensitivity = 80% and specificity = 67%.Sepsis represents a significant and growing challenge within modern healthcare settings. It can deteriorate quickly, potentially leading to fatality. The common characteristic to all sepsis patients is the importance of pathogen identification. Currently, pathogen detection is lengthy (hours to days) and as such is detrimental to patient outcome. Rapid pathogen detection will aid clinicians in source control and help guide targeted treatment in shorter time scales giving patients the best chances for recovery. Electrochemical biosensors have potential to offer a route to rapid, low cost, multiplexed pathogen identification. The work presented in this thesis represents a series of investigations made to apply electrochemical methods to rapid pathogen detection at the point of care. The first section presents a literature review exploring sepsis, current detection methods employed and identifying the requirements of an electrochemical biosensor for pathogen detection. The second section details investigations into suitable platforms on which to construct electrochemical biosensors. Various electrode platforms were investigated and the results from these experiments were used to design custom ‘printed circuit board’ electrodes. The ability to form sensing layers composed of biorecognition elements on printed circuit board electrode surfaces is then demonstrated using Streptococcus pneumoniae genomic DNA to form a S. pneumoniae recognition surface. lytA was detectable on this S. pneumoniae DNA biosensor in serum samples at 1 pM after 15 minutes of sample addition at room temperature. Finally, the development of a simple enzyme-based sensor for SARS-CoV-2 is demonstrated. Detection performances are described for multiple positive and negative controls culminating in clinical sample testing. Viral PCR sample solution clinical samples are shown to be statistically different from a negative sample (P = 1.2E-7). Viral transport medium clinical samples showed multiple positive and negative samples were significantly different with preliminary calculations showing a sensitivity = 80% and specificity = 67%

A Microelectrode Array with Reproducible Performance Shows Loss of Consistency Following Functionalization with a Self-Assembled 6-Mercapto-1-hexanol Layer

For analytical applications involving label free biosensors and multiple measurements, i.e. across an electrode array, it is essential to develop complete sensor systems capable of functionalisation and of producing highly consistent responses. To achieve this, a multi-microelectrode device bearing twenty-four equivalent 50 µm diameter Pt disc microelectrodes was designed in an integrated 3-electrode system configuration and then fabricated. Cyclic voltammetry and electrochemical impedance spectroscopy were used for initial electrochemical characterisation of the individual working electrodes. These confirmed the expected consistency of performance with a high degree of measurement reproducibility for each microelectrode across the array. With the aim of assessing the potential for production of an enhanced multi-electrode sensor for biomedical use, the working electrodes were then functionalised with 6-mercapto-1-hexanol (MCH). This is a well-known and commonly employed surface modification process, which involves the same principles of thiol attachment chemistry and self-assembled monolayer (SAM) formation commonly employed in the functionalisation of electrodes and the formation of biosensors. Following this SAM formation, the reproducibility of the observed electrochemical signal between electrodes was seen to decrease markedly, compromising the ability to achieve consistent analytical measurements from the sensor array following this relatively simple and well-established surface modification. To successfully and consistently functionalise the sensors it was necessary to dilute the constituent molecules by a factor of ten thousand to support adequate SAM formation on microelectrodes. The use of this multi-electrode device therefore demonstrates in a high throughput manner irreproducibility in the SAM formation process at the higher concentration, even though these electrodes are apparently functionalised simultaneously in the same film formation environment, confirming that the often seen significant electrode-to-electrode variation in label-free SAM biosensing films formed under such conditions is not likely to be due to variation in film deposition conditions, but rather kinetically controlled variation in the SAM layer formation process at these microelectrodes

Biologically modified microelectrode sensors provide enhanced sensitivity for detection of nucleic acid sequences from Mycobacterium tuberculosis

This paper describes improved sensitivity when using biosensors based on microfabricated microelectrodes to detect DNA, with the goal of progressing towards a low cost and mass manufacturable assay for antibiotic resistance in tuberculosis (TB). The microelectrodes gave a near 20 times improvement in sensitivity compared to polycrystalline macroelectrodes. In addition, experimental parameters such as redox mediator concentration and experimental technique were investigated and optimised. It was found that lower concentrations of redox mediator gave higher signal changes when measuring hybridisation events and, at these lower concentrations, square wave voltammetry was more sensitive and consistent than differential pulse voltammetry. Together, this paper presents a quantifiable comparison of macroelectrode and microelectrode DNA biosensors. The final assay demonstrates enhanced sensitivity through reduction of sensor size, reduction of redox mediator concentration and judicious choice of detection technique, therefore maintaining manufacturability for incorporation into point of care tests and lab-on-a-chip devices

Establishing a field-effect transistor sensor for the detection of mutations in the tumour protein 53 gene (TP53) : an electrochemical optimisation approach

We present a low-cost, sensitive and specific DNA field-effect transistor sensor for the rapid detection of a common mutation to the tumour protein 53 gene (TP53). The sensor consists of a commercially available, low-cost, field-effect transistor attached in series to a gold electrode sensing pad for DNA hybridisation. The sensor has been predominantly optimised electrochemically, particularly with respect to open circuit potentiometry as a route towards understanding potential (voltage) changes upon DNA hybridisation using a transistor. The developed sensor responds sensitively to TP53 mutant DNA as low as 100 nM concentration. The sensor responds linearly as a function of DNA target concentration and is able to differentiate between complementary and non-complementary DNA target sequences

An electrochemical SARS-CoV-2 biosensor inspired by glucose test strip manufacturing processes

Accurate and rapid diagnostic tests are critical to reducing the impact of SARS-CoV-2. This study presents early, but promising measurements of SARS-CoV-2 using the ACE2 enzyme as the recognition element to achieve clinically relevant detection. The test provides a scalable route to sensitive, specific, rapid and low cost mass testing

SARS-CoV-2 aptasensors based on electrochemical impedance spectroscopy and low-cost gold electrode substrates

SARS-CoV-2 diagnostic practices broadly involve either quantitative polymerase chain reaction (qPCR)-based nucleic amplification of viral sequences or antigen-based tests such as lateral flow assays (LFAs). Reverse transcriptase-qPCR can detect viral RNA and is the gold standard for sensitivity. However, the technique is time-consuming and requires expensive laboratory infrastructure and trained staff. LFAs are lower in cost and near real time, and because they are antigen-based, they have the potential to provide a more accurate indication of a disease state. However, LFAs are reported to have low real-world sensitivity and in most cases are only qualitative. Here, an antigen-based electrochemical aptamer sensor is presented, which has the potential to address some of these shortfalls. An aptamer, raised to the SARS-CoV-2 spike protein, was immobilized on a low-cost gold-coated polyester substrate adapted from the blood glucose testing industry. Clinically relevant detection levels for SARS-CoV-2 are achieved in a simple, label-free measurement format using sample incubation times as short as 15 min on nasopharyngeal swab samples. This assay can readily be optimized for mass manufacture and is compatible with a low-cost meter

An uncomplicated electrochemical sensor combining a perfluorocarbon SAM and ACE2 as the bio-recognition element to sensitively and specifically detect SARS-CoV-2 in complex samples.

Emerging in late 2019, the SARS-CoV-2 virus has had a devastating health and economic effects around the world forcing governments to enact restrictions on day to day life, resulting in severe economic and social disruption. The virus has stimulated new research in the fields of drug development, vaccinology and diagnostic testing. Here we present the basis for a simple, mass manufacturable saliva based electrochemical assay for the SARS-CoV-2 virus acheived through adsorption of the Angiotsnsin Converting Enzyme 2 (ACE2) into thiolated amphiphobic prefluoro monolayer assemled on a gold sensor surface. Following sensor preparation, it is possible to measure specific binding of recombinant spike protein and discriminate positive and negative samples of inactivated SARS-CoV-2 following 30 minutes incubation under ambient conditions. Representative calculations of limits of detection are made for recombinant spike protein (1.68 ng/ml) and inactivated virus (37.8 dC/mL). The assay as presented ultimately shows discrimination between positive and negative inactivated SARS-CoV-2 samples originating from clinical molecular standards kit intended for clinical and biomedical assay validation, and which is designed to mimic clinical samples through presence of cells and proteins in the sample medium. The simple design of the label free measurement and the selection of reagents involved means the assay has clear potential for transfer onto mass producible units such as screen-printed electrodes similar to glucose-format test strips, to enable widespread, low cost and rapid testing for SARS-CoV-2 in the general populatio

A SARS-CoV-2 aptasensor based on electrochemical impedance spectroscopy and low-cost gold electrode substrates.

SARS-CoV-2 diagnostic practices broadly involve either qPCR based nucleic amplification or lateral flow assays (LFAs). qPCR based techniques suffer from the disadvantage of requiring thermal cycling (difficult to implement for low-cost field use) leading to limitation on sample to answer time, the potential to amplify viral RNA sequences after a person is no longer infectious and being reagent intense. LFA performance is restricted by qualitative or semi-quantitative readouts, limits on sensitivity and poor reproducibility. Electrochemical biosensors, and particularly glucose test strips, present an appealing platform for development of biosensing solutions for SARS-CoV-2 as they can be multiplexed and implemented at very low cost at point of use with high sensitivity and quantitative digital readout. This work reports the successful raising of an Opti-mer sequence for the spike protein of SARS-CoV-2 and then development of an impedimetric biosensor which utilises thin film gold sensors on low-cost laminate substrates from home blood glucose monitoring. Clinically relevant detection levels for SARS-CoV-2 are achieved in a simple, label-free measurement format using sample incubation times of 15 minutes. The biosensor developed here is compatible with mass manufacture, is sensitive and low-cost CE marked readout instruments already exist. These findings pave the way to a low cost and mass manufacturable test with the potential to overcome the limitations associated with current technologies