Single-molecule detection of biomarkers for hepatocellular carcinoma and cholangiocarcinoma using novel nanopore-based sensing platforms
File(s)
Author(s)
Damiani, Micol
Type
Thesis
Abstract
Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), the most common types of liver malignancy, are characterised by a 5-year survival rate of 5%, mostly due late diagnosis. Since clear symptoms usually appear at advanced stages, conventional diagnostic approaches fail to detect the diseases at early stages. There is therefore a pressing need in both HCC and CCA for effective, reliable, non-invasive tools for early diagnosis, prognosis evaluation and at-risk-patients monitoring. Recent studies have identified panels of biomarkers (proteins, miRNAs) for both cancers. However, the presence of more abundant biomolecules hinders the detection of biomarkers in patient’s samples.
Nanopore sensing techniques, with their single-molecule resolution, offer powerful platforms to overcome these limitations. Being versatile, high-throughput and label-free, nanopores have been applied in the study of nucleic acids and proteins. However, their lack of selectivity hinders the utilization of nanopores in diagnostics.
This work presents two different strategies to detect miRNAs associated to CCA and HCC. The first combines nanopore-based electrical detection with fluorescent-based detection. Two molecular beacons, which bind two miRNAs upregulated in CCA, were hybridised to a DNA carrier, and the synchronised electro-optical signal originating from the target-bound carrier confirmed target binding.
The second platform combines DNA barcoded molecular probes and nanopore sequencing, to specifically detect 29 miRNAs associated with HCC or CCA. We demonstrated that the custom-designed probes enable the multiplexed detection and quantification of the 29 synthetic nucleic acid targets. The platform was then optimised for the use of human serum. Preliminary experiments were performed using clinical samples. While certain miRNAs were successfully detected, no significant difference was found between healthy controls and cancer serum samples. Further optimisation and control experiments are therefore required. Overall, this method could represent the first step towards the development of a portable and innovative non-invasive diagnostic and prognostic tool for CCA and HCC.
Nanopore sensing techniques, with their single-molecule resolution, offer powerful platforms to overcome these limitations. Being versatile, high-throughput and label-free, nanopores have been applied in the study of nucleic acids and proteins. However, their lack of selectivity hinders the utilization of nanopores in diagnostics.
This work presents two different strategies to detect miRNAs associated to CCA and HCC. The first combines nanopore-based electrical detection with fluorescent-based detection. Two molecular beacons, which bind two miRNAs upregulated in CCA, were hybridised to a DNA carrier, and the synchronised electro-optical signal originating from the target-bound carrier confirmed target binding.
The second platform combines DNA barcoded molecular probes and nanopore sequencing, to specifically detect 29 miRNAs associated with HCC or CCA. We demonstrated that the custom-designed probes enable the multiplexed detection and quantification of the 29 synthetic nucleic acid targets. The platform was then optimised for the use of human serum. Preliminary experiments were performed using clinical samples. While certain miRNAs were successfully detected, no significant difference was found between healthy controls and cancer serum samples. Further optimisation and control experiments are therefore required. Overall, this method could represent the first step towards the development of a portable and innovative non-invasive diagnostic and prognostic tool for CCA and HCC.
Version
Open Access
Date Issued
2023-12-12
Date Awarded
2024-04-01
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Edel, Joshua
Ivanov, Aleksandar
Khan, Shahid
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)