Modified nanotweezers for subcellular extraction of cytoplasmic mrna
File(s)
Author(s)
Monteza Cabrejos, Tony
Type
Thesis
Abstract
This thesis describes the development and application of modified dielectrophoretic nanotweezers designed to extract mRNA from living cells, offering a novel method for analysing transcriptional changes in luminal breast cancer cells exhibiting treatment resistance. The enhanced nanotweezers leverage dielectrophoresis to selectively trap biomolecules without aspirating cytoplasmic fluid, thereby maintaining cell viability during sampling.
A key modification of the nanotweezers involved incorporating a poly-thymine oligomer on the device's surface, leading to a marked increase in mRNA trapping capacity from single-cell biopsies. This enabled the extraction of a continuous dataset of truly longitudinal transcriptional profiles from individual breast cancer cells over 28 days, providing insights into the cellular response to endocrine therapies and the emergence of dormancy and resistance patterns that could potentially lead to metastasis.
Further developments in nanotweezer technology were explored, and specific primer sequences were added to target low-abundance transcripts. This selectivity enhancement is essential for detailed transcriptomic analysis, allowing for more precise profiling of cellular processes at the molecular level.
The thesis also explores integrating nanotweezers with nanoparticles to create a reusable extraction system. This reduces the per-unit cost of extraction and ensures greater consistency between samples. Testing this system demonstrated its potential to be a sustainable tool in transcriptomic studies, providing a means to sample single cells over time. However, work is still needed to optimise the system for reusable extraction.
The modifications and applications of the nanotweezers detailed in this thesis contribute to the field of single-cell transcriptomics. They demonstrate the potential for these tools to provide a more detailed understanding of cellular responses to treatment, which is critical for advancing breast cancer research and developing more effective therapeutic strategies. The refined technology presents a versatile platform for addressing complex questions in cellular biology, with potential for broad application in biomedical research, such as personalised medicine.
A key modification of the nanotweezers involved incorporating a poly-thymine oligomer on the device's surface, leading to a marked increase in mRNA trapping capacity from single-cell biopsies. This enabled the extraction of a continuous dataset of truly longitudinal transcriptional profiles from individual breast cancer cells over 28 days, providing insights into the cellular response to endocrine therapies and the emergence of dormancy and resistance patterns that could potentially lead to metastasis.
Further developments in nanotweezer technology were explored, and specific primer sequences were added to target low-abundance transcripts. This selectivity enhancement is essential for detailed transcriptomic analysis, allowing for more precise profiling of cellular processes at the molecular level.
The thesis also explores integrating nanotweezers with nanoparticles to create a reusable extraction system. This reduces the per-unit cost of extraction and ensures greater consistency between samples. Testing this system demonstrated its potential to be a sustainable tool in transcriptomic studies, providing a means to sample single cells over time. However, work is still needed to optimise the system for reusable extraction.
The modifications and applications of the nanotweezers detailed in this thesis contribute to the field of single-cell transcriptomics. They demonstrate the potential for these tools to provide a more detailed understanding of cellular responses to treatment, which is critical for advancing breast cancer research and developing more effective therapeutic strategies. The refined technology presents a versatile platform for addressing complex questions in cellular biology, with potential for broad application in biomedical research, such as personalised medicine.
Version
Open Access
Date Issued
2023-11-30
Date Awarded
01/08/2024
License URL
Advisor
Edel, Joshua
Ivanov, Aleksandr
Magnani, Luca
Sponsor
Engineering and Physical Sciences Research Council
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)