The selective and sensitive detection of protein biomarkers using aptamer-based nanosensors
File(s)
Author(s)
Edwards, Benjamin James
Type
Thesis
Abstract
A multitude of diseases are known to have specific biomarkers yet detection with high specificity and selectivity remains highly challenging. For example, single molecule-based fluorescence techniques while highly sensitive, requires labelling with expensive fluorescent tags, as well as recognition elements (e.g. antibodies) if detection is to be carried out in complex solutions. Such labelling compromises the biophysical properties of the analyte likely resulting in altered aggregation. To address these limitations, the PhD project aimed to combine the single molecule and label-free detection granted by nanopores with highly specific single stranded oligonucleotides known as aptamers in order to detect biologically relevant proteins.
The aptamer-based nanosensor was constructed in a two-step method. An initial silanisation step carried out in the vapour phase allowed the formation of a mono-silane layer with a terminal methacrylate functional group, which in turn allowed a thiol-modified aptamer to be added via click chemistry-based Michael addition. Characterisation of the developed sensor was carried out electrochemically via in-situ I-V curves, while further confirmation could be seen through the presence of substantial current blockades upon addition of complementary (to the aptamer) nucleotide sequences.
The primary aim of the project was to detect thrombin, a biomarker which has links to Multiple Sclerosis and cancer. Thrombin provided an excellent initial target due to its well characterised structure alongside a high affinity (kD = 0.5 nM) binding aptamer. Characterisation of the developed sensor was initially carried out via concentration and voltage-based assays, before showing that the aptamer-based nanosensor could not only be regenerated but also carried out in multi-protein solutions. In addition, highly cytotoxic alpha synuclein oligomers; the likely biomarkers of Parkinson’s Disease have been detected. After a concentration-based assay, it was shown that the (anti-oligomer) aptamer specifically targeted a specific oligomer sub-type when samples were detected over a range of different aggregation timepoints.
Overall the developed aptamer-based nanosensor is able to specifically detect high-value biomarkers at ultra-low concentrations, underlying its potential to be used in the healthcare industry.
The aptamer-based nanosensor was constructed in a two-step method. An initial silanisation step carried out in the vapour phase allowed the formation of a mono-silane layer with a terminal methacrylate functional group, which in turn allowed a thiol-modified aptamer to be added via click chemistry-based Michael addition. Characterisation of the developed sensor was carried out electrochemically via in-situ I-V curves, while further confirmation could be seen through the presence of substantial current blockades upon addition of complementary (to the aptamer) nucleotide sequences.
The primary aim of the project was to detect thrombin, a biomarker which has links to Multiple Sclerosis and cancer. Thrombin provided an excellent initial target due to its well characterised structure alongside a high affinity (kD = 0.5 nM) binding aptamer. Characterisation of the developed sensor was initially carried out via concentration and voltage-based assays, before showing that the aptamer-based nanosensor could not only be regenerated but also carried out in multi-protein solutions. In addition, highly cytotoxic alpha synuclein oligomers; the likely biomarkers of Parkinson’s Disease have been detected. After a concentration-based assay, it was shown that the (anti-oligomer) aptamer specifically targeted a specific oligomer sub-type when samples were detected over a range of different aggregation timepoints.
Overall the developed aptamer-based nanosensor is able to specifically detect high-value biomarkers at ultra-low concentrations, underlying its potential to be used in the healthcare industry.
Version
Open Access
Date Issued
2020-06
Date Awarded
2020-10
Copyright Statement
Creative Commons Attribution Non-Commercial NoDerivatives Licence
Advisor
Ivanov, Alexander
Edel, Joshua
Cass, Anthony
Sponsor
Imperial College London
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)