A 3D Microfluidic Chip for Electrochemical Detection of Hydrolysed Nucleic Bases by a Modified Glassy Carbon Electrode

Modification of carbon materials, especially graphene-based materials, has wide applications in electrochemical detection such as electrochemical lab-on-chip devices. A glassy carbon electrode (GCE) modified with chemically alternated graphene oxide was used as a working electrode (glassy carbon modified by graphene oxide with sulphur containing compounds and Nafion) for detection of nucleobases in hydrolysed samples (HCl pH = 2.9, 100 °C, 1 h, neutralization by NaOH). It was found out that modification, especially with trithiocyanuric acid, increased the sensitivity of detection in comparison with pure GCE. All processes were finally implemented in a microfluidic chip formed with a 3D printer by fused deposition modelling technology. As a material for chip fabrication, acrylonitrile butadiene styrene was chosen because of its mechanical and chemical stability. The chip contained the one chamber for the hydrolysis of the nucleic acid and another for the electrochemical detection by the modified GCE. This chamber was fabricated to allow for replacement of the GCE

Low-cost pH sensors based on discrete PCB ion-sensitive field effect transistors

Diagnostic technologies will play a critical role in addressing current and future healthcare challenges, with the greatest impact through implementing these technologies at the point of care (PoC). For truly widespread deployment, these PoC technologies should be low-cost and amenable for mass manufacture, even in resource-limited settings, without compromising analytical performance. Discrete, extended gate pH-sensitive field-effect transistors (dEGFETs) fabricated on widely used printed circuit boards (PCBs) are a low-cost, simple to manufacture analytical technology. Electrodeposited iridium oxide (IrOx) films have emerged as a promising pH-sensitive transducer due to their facile deposition. While IrOx is predicted to have a beyond-Nernstian pH sensitivity, the performance measured experimentally is typically lower and variable. This thesis demonstrates a dEGFET pH sensor based on PCB extended gate electrodes and electrodeposited IrOx, which repeatedly displays beyond-Nernstian pH response. Using complementary surface-analysis techniques, it is shown the high pH sensitivity and repeatability is determined both by the chemical composition and critically the uniformity of the IrOx film. Electrochemical polishing of the extended gate electrode prior to electrodeposition enhances IrOx uniformity, leading to a median pH sensitivity of 70.7 ± 5 mV/pH (n=56) compared to 31.3 ± 14 mV/pH (n=31) for non-polished electrodes. The applicability of these devices is demonstrated through the quantification of the β-lactam antibiotic ampicillin, via the pH change that occurs due to hydrolysis catalysed by β-lactamase enzymes. This lays the foundations for a susceptibility assay towards the public health challenge of antimicrobial resistance (AMR). Additionally, this thesis explores the integration of electronically controlled microfluidic valves onto the PCB substrates, towards the development of lab-on-chip systems and PoC diagnostics. The highly sensitive and repeatable dEGFET sensors presented here show great promise as a low-cost diagnostic technology. Moreover, the use of PCB substrates is suitable for manufacture in resource-limited settings, enabling widespread diagnostic testing

Fundamental studies towards the fabrication of electroactive monolithic stationary phases in microfluidic channels

The long term goal of this project is to develop a monolithic stationary phase which utilises an electroactive polymer combining the advantages of EMLC, monolithic technology and microfluidic separation, thus creating an electroactive monolithic microchip (EMμ). In this thesis, fundamental studies towards the fabrication of EMμ are presented, i.e. integration of an electrochemical cell into a microfluidic chip, colloidal crystallization in microfluidic channels and PANI growth through a colloidal crystal template. Polyaniline was selected as the electroactive material for the fabrication of the monolithic stationary phase as its use for EMLC had already been demonstrated. Colloidal crystals have been used to microstructure materials and the inverse opal structure comprises pore sizes of the order of what was needed for EMμ; therefore electropolymerization of aniline through a polystyrene colloidal crystal template strategy was chosen. Two alternative chip designs, CD1 and CD2, were investigated for this thesis. Their applicability for EMμ was assessed in terms of their flow velocity profile using computational fluid dynamic, colloidal crystallization feasibility and electrochemical behavior using ferricyanide electrochemistry. The integration of a fully operational three-electrode electrochemical cell within a microfluidic channel and its use for polyaniline electropolymerization was demonstrated, and self-assembly of the sacrificial polystyrene template in these channels was shown. Polyaniline microstructure morphology exhibited a dependence on the surfactant concentration present in the polystyrene suspension. Finally, electrochemical switching of conducting polymer within microfluidic channels was assessed by studying polypyrrole switching by atomic force microscopy (AFM). Pore swelling and contraction was observed on application of a potential, demonstrating that the monolith properties could be dynamically controlled. It was found that volume increase in the polymer could be responsible for a deformation of flow through pores due to physical confinement of the polymer

Development of electrochemical DNA-based biosensors for the detection of Shiga toxin-producing E. coli (STEC)

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 ix 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 x 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

Overview of optical and electrochemical alkaline phosphatase (ALP) biosensors: recent approaches in cells culture techniques

Alkaline phosphatase (ALP), which catalyzes the dephosphorylation process of proteins, nucleic acids, and small molecules, can be found in a variety of tissues (intestine, liver, bone, kidney, and placenta) of almost all living organisms. This enzyme has been extensively used as a biomarker in enzyme immunoassays and molecular biology. ALP is also one of the most commonly assayed enzymes in routine clinical practice. Due to its close relation to a variety of pathological processes, ALP’s abnormal level is an important diagnostic biomarker of many human diseases, such as liver dysfunction, bone diseases, kidney acute injury, and cancer. Therefore, the development of convenient and reliable assay methods for monitoring ALP activity/level is extremely important and valuable, not only for clinical diagnoses but also in the area of biomedical research. This paper comprehensively reviews the strategies of optical and electrochemical detection of ALP and discusses the electrochemical techniques that have been addressed to make them suitable for ALP analysis in cell culture

Electrochemical cellular and nucleic Acid based biomarker detection of neuroblastoma.

The rapid, specific, and sensitive detection of biomarkers for disease state and progression has become an expansive field in research due to advances in both the understanding of biomarkers and diagnostic detection strategies. The detection of both cellular and molecular biomarkers through electrochemical means provides a rapid and sensitive strategy for the detection of specific markers of disease, in this case neuroblastoma. Chapter 1 of this thesis reviews the current literature reports on both cellular and molecular biomarkers for neuroblastoma disease progression as well as detection methods, both electrochemical and otherwise, that may be applied to the detection of neuroblastoma. In Chapter 2 the detection of cellular biomarkers of neuroblastoma via electrochemical analysis is discussed, chiefly focusing on the immobilisation of neuroblastoma cells to an electrode surface and the subsequent detection of immobilised cells through electrochemical impedance spectroscopy and amperometric detection of electrocatalytic platinum nanoparticles. Following on from this, Chapter 3 focuses on the detection of a molecular biomarker of neuroblastoma disease progression, miR-132. This detection strategy relies on the partial hybridisation of the miRNA target, miR-132, to a self-assembled monolayer of nucleic acid strands on the surface of an electrode. Electrocatalytic platinum nanoparticles uniformly decorated with probe oligonucleotides are then hybridised to the free end of the miRNA target and detected amperometrically through the electrocatalytic reduction of H2O2 at the nanoparticle surface. The focus of this work then shifts in Chapter 4 to the formation of a detection strategy based on electrochemiluminescence, whereby a luminophore, Ruthenium II (bis-2,2-bipyridyl)-2(4-aminophenyl) imidazo[4,5-f][1,10] phenanthroline is immobilised to the surface of a nanoparticle decorated electrode surface and is detected by ECL. This body of work explores possible avenues for the enhancement of the electrochemiluminescent signal generated by immobilised luminophore by plasmonic enhancement of luminescent signal through nanotexturing of electrode surfaces. Chapter 4 then details work carried out to translate this work to a working assay for the detection of miRNA using multiple combinations of nanoparticle (Au/Ag/Pt) and luminophore. By carefully selecting the electrode surface and luminophore an overall enhancement of signal is observed for immobilised probes and a calibration plot for the concentration of miR-132 applied to the electrode surface can be obtained

Effects of stratospheric conditions on viability, metabolism and proteome of prokaryotic cells

The application of ultraviolet (UV) radiation to inhibit bacterial growth is based on the principle that the exposure of DNA to UV radiation results in the formation of cytotoxic lesions, leading to inactivation of microorganisms. Herein, we present the impacts of UV radiation on bacterial cultures' properties from the biological, biochemical and molecular biological perspective. For experiments, commercial bacterial cultures (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli and Salmonella typhimurium) and isolates from patients with bacterial infections (Proteus mirabilis and Pseudomonas aeruginosa) were employed. The above-mentioned strains were exposed to UV using a laboratory source and to stratospheric UV using a 3D printed probe carried by a stratospheric balloon. The length of flight was approximately two hours, and the probe was enriched by sensors for the external environment (temperature, pressure and relative humidity). After the landing, bacterial cultures were cultivated immediately. Experimental results showed a significant effect of UV radiation (both laboratory UV and UV from the stratosphere) on the growth, reproduction, behavior and structure of bacterial cultures. In all parts of the experiment, UV from the stratosphere showed stronger effects when compared to the effects of laboratory UV. The growth of bacteria was inhibited by more than 50% in all cases; moreover, in the case of P. aeruginosa, the growth was even totally inhibited. Due to the effect of UV radiation, an increased susceptibility of bacterial strains to environmental influences was also observed. By using commercial tests for biochemical markers of Gram-positive and Gram-negative strains, significant disparities in exposed and non-exposed strains were found. Protein patterns obtained using MALDI-TOF mass spectrometry revealed that UV exposure is able to affect the proteins' expression, leading to their downregulation, observed as the disappearance of their peaks from the mass spectrum

Quantum dots genosensor for Her2/Neu oncogene - a breast cancer biomarker

Philosophiae Doctor - PhDThe human epidermal growth factor receptor (HER)-family of receptor tyrosine kinases; human epidermal growth factor receptor 1, human epidermal growth factor receptor 2, human epidermal growth factor receptor 3 and human epidermal growth factor receptor 4 (EGFR/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4) plays a major role in the pathogenesis of many solid tumours, in approximately 25 - 30% of breast cancers. Breast cancer is the second most common type of cancer and affects around 3000 women annually in South Africa alone. While the benefits of treatment and cancer progress to enhance therapeutic effectiveness for the patient are well documented, it is also important to employ or fabricate methods in which cancer can be screened at an early stage. A number of gene and protein based biomarkers have shown potential in the early screening of cancer. One specific biomarker that is over-expressed in 20 - 30% of human breast cancers is the human epidermal growth factor receptor 2 (Her2/neu). Several methods have been developed for detection of Her2/neu oncogene including immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), fluorescent in situ hybridisation (FISH) and polymerase chain reaction(PCR). However, these methods are subjected to interference problem. For these reasons an ultrasensitive, cheap and easy to use genosensor has been developed for early detection of the Her2/neu oncogene using electrochemical and spectroscopic methods. Due to their high surface-to-volume ratio, electro-catalytic activity as well as good biocompatibility and novel electron transport properties quantum dots are highly attractive materials for ultra-sensitive detection of biological macromolecules via bio-electronic or bio-optic devices. In this study a quantum dots (QDs)-based genosensor was developed in which Ga2Te3-based quantum dots were synthesised using a novel aqueous solution approach by mixing 3-mercaptopropionic acid (3MPA)-capped gallium metal precursor with reduced tellurium metal. The morphological, compositional and structural characterisation of the QDs was investigated prior to their utilization in DNA sensor construction