Multiplexed detection of microRNAs, proteins and neurotransmitter using barcoded molecular probes and nanopore sequencing
File(s)
Author(s)
Koch, Caroline
Type
Thesis
Abstract
In the field of clinical diagnostics, the identification of biomarker concentrations associated with specific diseases is critical. Typically, a limited set of biomarkers is examined, frequently falling short in delivering comprehensive clinical detail on the pathology and consequently leading to misdiagnoses and subsequent over- or under-treatment of patients.
Conventional techniques like RT-qPCR and ELISA fall short in the simultaneous detection of various biomarker classes, such as microRNAs, proteins, and small molecules, necessitating a novel diagnostic approach for multiplexed biomarker detection. To overcome this challenge, we developed a platform to detect and differentiate a diverse panel of biomarkers within complex biological fluids. We designed barcoded molecular probes that selectively bind to different biomarkers including microRNAs, proteins, and small molecules, and combined their usage with nanopore sequencing to enable high throughput.
Initial experiments showcased successful multiplexed detection and quantification of 40 synthetic miRNAs using custom-designed barcoded molecular probes, enabling blinded studies to ascertain unknown miRNA concentrations. Furthermore, individual proteins were detected, and proof-of-concept experiments showcased the multiplexed detection of microRNAs, proteins, and small molecules. This multiplexed approach was validated in clinical samples, demonstrating the simultaneous detection of 40 different microRNAs in healthy participants, as well as in patients suffering from cardiac disease.
Our approach offers adaptability, allowing for easy customization of target analytes and numbers, and facilitating personalized detection strategies. These advancements signify promising strides towards developing a portable, non-invasive diagnostic tool, with the potential to enhance disease management and patient monitoring upon further optimization.
Conventional techniques like RT-qPCR and ELISA fall short in the simultaneous detection of various biomarker classes, such as microRNAs, proteins, and small molecules, necessitating a novel diagnostic approach for multiplexed biomarker detection. To overcome this challenge, we developed a platform to detect and differentiate a diverse panel of biomarkers within complex biological fluids. We designed barcoded molecular probes that selectively bind to different biomarkers including microRNAs, proteins, and small molecules, and combined their usage with nanopore sequencing to enable high throughput.
Initial experiments showcased successful multiplexed detection and quantification of 40 synthetic miRNAs using custom-designed barcoded molecular probes, enabling blinded studies to ascertain unknown miRNA concentrations. Furthermore, individual proteins were detected, and proof-of-concept experiments showcased the multiplexed detection of microRNAs, proteins, and small molecules. This multiplexed approach was validated in clinical samples, demonstrating the simultaneous detection of 40 different microRNAs in healthy participants, as well as in patients suffering from cardiac disease.
Our approach offers adaptability, allowing for easy customization of target analytes and numbers, and facilitating personalized detection strategies. These advancements signify promising strides towards developing a portable, non-invasive diagnostic tool, with the potential to enhance disease management and patient monitoring upon further optimization.
Version
Open Access
Date Issued
2024-03-19
Date Awarded
2024-06-01
Copyright Statement
Attribution-NonCommercial 4.0 International Licence (CC BY-NC)
License URL
Advisor
Edel, Joshua
Ivanov, Aleksandar
Gutierrez, Richard
Bruce, Mark
Sponsor
Oxford Nanopore Technologies
Engineering and Physical Sciences Research Council
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)