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Tan, Shi Hui (2025) Envelope glycoprotein domain III (EDIII): potential as bio-receptor probe for specific detection of different serotypes of dengue antibodies. PhD thesis, University of Nottingham.

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Abstract

Dengue fever has been a global threatening disease, affecting approximately 40% of the world’s population (3.6 billion) residing in dengue-endemic areas across more than 100 countries within the World Health Organisation (WHO) regions. Dengue fever is caused by dengue virus (DENV) which constitutes four distinct antigenic serotypes (DENV1, DENV2, DENV3 and DENV4). Infection with one serotype of DENV is thought to produce long-term immunity against that particular serotype but only short-term cross-immunity against the other serotypes. Sequential infections with different DENV serotypes lead to cross-reactive but also serotype-specific neutralizing antibody responses. This potentially results in life-threatening complications, i.e. antibody dependent enhancement (ADE). ADE is a phenomenon in which the antibodies produced during a previous immune response recognize and bind to the pathogen, but they are unable to prevent the subsequent infection. Instead, they facilitate viral entry into cells, leading to increased viral replication and more severe disease outcomes. Therefore, there is a pressing need to develop a cheap, rapid, and highly sensitive point-of-care diagnostic tool for early serotype-specific DENV detection, as early detection and prompt medical treatment can reduce the mortality rates associated with dengue fever. However, the current diagnostic landscape lacks sufficiently sensitive, specific, and rapid tools capable of differentiating DENV infections based on serotypes.

Electrochemical biosensor serves an important role in the advancement of commercial point-of-care systems, to detect the presence of various biological analytes or targets, such as glucose, uric acid, and cholesterol for diagnostic purposes. Electrochemical biosensor is an analytical device which converts a biological response into a quantifiable and processable signal using electrochemistry. This approach is currently dominating the biosensing field with the common techniques, such as amperometry, cyclic voltammetry, direct pulse voltammetry and electrical impedance spectroscopy. A key aspect of biosensor development involves selecting a suitable antigen probe for specific and sensitive detection. In this study, a consensus DENV envelope domain III (cEDIII) protein probe was employed due to its high potential for recognising and binding antibodies generated against different DENV serotypes, making it a valuable component for future development of a serotype-specific diagnostic tool. Leveraging this probe, a nanomaterial-based electrochemical biosensor was proposed as a reliable cost-effective diagnostic tool for dengue fever with the overarching aim of detecting different DENV serotypes with rapid results, high selectivity, and specificity.

A graphene/titanium dioxide-based electrochemical biosensor had been successfully developed in specifically detecting three different targets, namely DENV1-4 immunoglobin G (IgG), DENV1 IgG and DENV2 IgG, by using the plant-derived cEDIII with cholera toxin B (CTB) fusion (CTB-cEDIII) as the antigen probe. CTB was fused with cEDIII to enhance overall structural stability by providing a stable pentameric scaffold. The immunosensor exhibited high sensitivity and selectivity towards DENV1-4 IgG, DENV1 IgG and DENV2 IgG with the limits of detection of 12.17 ng/mL, 14.05 ng/mL, and 10.56 ng/mL, respectively. The linear working ranges for DENV1-4 IgG, DENV1 IgG and DENV2 IgG were 31.25 – 1,000 ng/mL, 562.5 – 9,000 ng/mL and 31.25 – 500 ng/mL, with limits of quantification of 36.87 ng/mL, 42.49 ng/mL, and 31.87 ng/mL, respectively. The as-developed immunosensor also demonstrated excellent selectivity against other closely related viral antibodies, such as Zika, Yellow Fever and Chikungunya. Moreover, the analytical performance of CTB-cEDIII as detection probe in biosensor platform was validated by the well-established conventional detection platform, i.e., enzyme-linked immunosorbent assay (ELISA). Biosensor was proved to be more advantageous than ELISA platform, offering rapid detection, lower cost, and ease of use.

Subsequently, the study advanced to the expression of a partially soluble bacterial based recombinant protein probe, CTB-cEDIII and cEDIII, utilising Escherichia coli (E. coli) host cells and the IMPACT-TWIN (Intein Mediated Purification with an Affinity Chitin binding Tag-Two Intein) system. This was due to the advantages of bacterial platform in terms of cost and time efficiency, and ease in post purification processes as compared to plant-based production. The molecular cloning and protein expression processes comprised several key stages, each significantly impacting its overall success, efficiency, and yield. Optimisation studies were carried out to understand the effect of key factors, such as the concentration and purity of cloning vectors and plasmid inserts, protein expression and extraction parameters. These parameters encompassed freeze-thaw cycles, isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration, induction temperature and duration. The parameters that yielded the optimum expression were freeze-thaw condition of 1 hour at -80°C and induction with 0.1 mM IPTG at 15°C overnight. The influence of protein structure in the purification step, involving chitin resin chromatography and ion exchange chromatography, was found to significantly impact the success rate of protein purification. It is essential to consider the downstream applications of the expressed DENV envelope protein via bacterial host, as high expression levels may not always correspond to functional activities. Nevertheless, the antigenicity of the bacterial-produced CTB-cEDIII protein was confirmed with sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western and dot blotting techniques. The protein sizes for semi-soluble recombinant proteins, CTB-cEDIII and cEDIII, were as expected at 25.5 kDa and 11.3 kDa, respectively.

In conclusion, the electrochemical immunosensor platform has been successfully developed for detecting DENV antibodies, with a specific focus on DENV serotype 1 and serotype 2. Although the successful expression of bacterial CTB-cEDIII protein represents a significant achievement, further refinement of purification processes holds the potential to improve the suitability of this bacterial CTB-cEDIII protein probe for the electrochemical DENV immunosensor platform, thereby advancing point-of-care dengue diagnostics in the future, including dengue serotype differentiation via post-analysis of immunosensor data using artificial intelligence.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Loh, Sandy Hwei San Tan, Michelle Tien Tien
Keywords:	dengue fever; global threat; endemic areas; antibody-dependent enhancement (ADE); serotype-specific detection
Subjects:	Q Science > QR Microbiology
Faculties/Schools:	University of Nottingham, Malaysia > Faculty of Science and Engineering — Science > School of Biosciences
Item ID:	80320
Depositing User:	Tan, Shi Hui
Date Deposited:	08 Feb 2025 04:40
Last Modified:	08 Feb 2025 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/80320

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