A customisable biosensor platform for point-of-care applications in precision medicine and immunodiagnostics
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2020-09-24.
© 2018 Dr Gita Soraya
Point-of-care diagnostics play an indispensable role in clinical decision-making pathways as rapid and cost-effective alternatives or complementation to laboratory- based testing. The overarching aim of this thesis was to develop a point-of-care biosensor platform for precision medicine and immunodiagnostic applications. Four sub-studies were undertaken to demonstrate sensor development within the two themes. The interdigitated electrode (IDE) biosensor was used as the core technology, based on its rapid and label-free potential, low-cost fabrication, and amenability towards integration into a compact lab-on-chip device. The first theme focused on biosensor development for pharmacogenetic testing. Associations between HLA variations and medication-specific adverse drug reactions (ADRs) have led to screening recommendations for the HLA-B*15:02 allele before carbamazepine therapy and the HLA-B*57:01 allele before abacavir therapy. However, implementation is hindered by the high-cost, complexity, and lengthy turnaround times of current gold standards. Sub-study 1 aimed to develop the IDE sensor for rapid genotyping of HLA-B*15:02, which is strongly associated with severe cutaneous reactions to the antiepileptic drug carbamazepine in broad Asian populations. A two-step detection platform was established, combining loop-mediated isothermal amplification (LAMP) of HLA-B alleles with HLA-B*15:02 LAMP amplicon hybridisation on the IDE sensor. Validation was performed on purified extracted DNA samples and 27 whole-blood samples from Hong-Kong, where HLA-B*15:02 is prevalent. Impedance characterisation of the LAMP-IDE platform resulted in 92.86% sensitivity and 84.63% specificity towards ii HLA-B*15:02, with a short turnaround time of ~1 hour and 20 minutes without additional DNA staining. Sub-study 2 aimed to develop a biosensor platform for typing of HLA-B*57:01, which is strongly associated with abacavir hypersensitivity A cell capture approach was established using a monoclonal antibody (3E12) specific towards the HLA-B17 group (encompassing HLA-B57 and HLA-B58). Detection of HLA-B*57:01 cell line and HLA-B57 peripheral blood mononuclear cell (PBMC) capture was achieved within 15 minutes. Impedance characterisation demonstrated differentiation between sensors incubated in HLA-B57 PBMCs with non-HLA-B57 PBMCs (p < 0.0001) and blank media (p = 0.02). The platform is also potentially applicable for prevention of allopurinol hypersensitivity through HLA-B*58:01 typing. The second theme explored the use of IDE sensors as an alternative method to bridge the gap between highly-sensitive benchtop immunoassays and less-sensitive and qualitative first-generation point-of-care immunoassays. Sub-study 3 demonstrated the development of a quantitative immunodiagnostic platform for colorectal cancer (CRC) screening. Using anti-haemoglobin (Hb) antibodies, the IDE sensor showed successful quantification of faecal occult blood within ~1 hour, with a sensitivity of 10 μg haemoglobin per gram (Hb/g) of faeces, comparable to laboratory-based quantitative faecal immunochemical testing (FIT) systems. In sub-study 4, an ultrasensitive IDE detection platform was developed for malaria elimination purposes, targeting the Plasmodium falciparum histidine-rich protein II (PfHRP2). Using anti-PfHRP2 antibodies, the IDE sensor successfully detected PfHRP2 at 2.5 pg/mL in PBS and 25 pg/mL in saliva within ~2 hours without labelling. iii Collectively, results of the four sub-studies demonstrate the IDE sensor’s versatility as a relatively-rapid, label-free, and customisable diagnostic platform, comparable to each application’s laboratory-based gold standard methods. The platform has potential to be integrated into a low-cost and compact lab-on-chip device for multiple diagnostic applications.
Keywordsbiosensor; biotechnology; diagnostics; point-of-care; interdigitated electrode; precision medicine; pharmacogenetics; human leukocyte antigen; malaria; immunodiagnostics; plasmodium falciparum histidine rich protein II; colorectal cancer
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- Medicine (RMH) - Theses