Nanotechnology platforms for detection and analysis of clinically relevant biological nanoparticles
The goal of this project was to develop platforms for the detection and analysis of extracellular vesicles (EVs). Detection, because it was hypothesized that the presence and concentration of various kinds of extracellular vesicles in patient blood samples, as measured in a liquid biopsy, would prove to be a valuable parameter to guide therapy of primarily cancer patients.Analysis, because despite the exponential growth of the field in recent years, there remain many mysteries regarding the biogenesis, composition and physiological behavior of these submicroscopic biological information carriers. In addition, and notwithstanding clear guidelines by expert societies, there is still much confusion in the literature about how to interpret experimental results – e.g., how to properly prepare samples or when to call a detected object an EV. The work described in this thesis aims to support the clinical context by providing technological concepts that could be useful both for fundamental biological studies into the occurrence and behavior of EVs and by giving considerations for sample preparation. It explains why extremely sensitive biosensors are required and describes the development of some lab-on-a-chip solutions.Chapter 1 introduces the concept of liquid biopsy and circulating tumor cells and explains the reasons to explore the feasibility of using EVs instead. It then goes deeper into the composition of EVs and their distribution in the blood. Next, challenges involved in sample preparation are discussed, as well as the barriers to high-throughput reliable detection of EVs.Chapter 2 gives an overview of the Cancer-ID consortium that provided the framework for this (sub)project. It highlights the findings of colleagues in Amsterdam, Delft, Groningen, Twente and Utrecht, who studied EVs from various perspectives in parallel. It gives a comprehensive comparison of the efficacy and throughput of various methods to study EVs for their corresponding applications.Chapter 3 explains the necessity for multi-modal analysis and introduces a first platform that allowed studying individual tumor-derived extracellular vesicles (tdEVs) with scanning electron microscopy, Raman spectrometry and atomic force microscopy to correlate the respectively morphological, biochemical and mechanical data obtained from these single nanoparticles.Chapter 4 reports observations made while performing sample preparation of EVs for different purposes. It was found that a class of polymers, i.e., organosilicon compounds, have a stable interaction with phospholipid membranes found in cells, EVs and artificial membrane models. This has implications for the use of these polymers not only in research but even in daily life, as these silicones occur in cosmetics, pharmaceutics and even food.Chapter 5 presents an integrated system that uses a two-stage identification of tdEVs followed by a two-stage signal amplification. This awarded a highly specific detection method: only tdEVs gave a detectable signal. It also proved to be ultrasensitive; thanks to miniaturization tot the nanoscale of an ELISA-like electrochemical detection mechanism, tdEVs were detectable at concentrations as low as 10 tdEVs/μl.Chapter 6 explores the qualities of a hypothetically ideal biosensor system and provides those solutions that are technologically available to us now.The last chapter discusses how the preceding chapters were able to answer questions existing in the field and what is necessary to further explore the remaining mysteries.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
| Published: |
Wageningen University
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| Subjects: | Life Science, |
| Online Access: | https://research.wur.nl/en/publications/nanotechnology-platforms-for-detection-and-analysis-of-clinically |
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| Summary: | The goal of this project was to develop platforms for the detection and analysis of extracellular vesicles (EVs). Detection, because it was hypothesized that the presence and concentration of various kinds of extracellular vesicles in patient blood samples, as measured in a liquid biopsy, would prove to be a valuable parameter to guide therapy of primarily cancer patients.Analysis, because despite the exponential growth of the field in recent years, there remain many mysteries regarding the biogenesis, composition and physiological behavior of these submicroscopic biological information carriers. In addition, and notwithstanding clear guidelines by expert societies, there is still much confusion in the literature about how to interpret experimental results – e.g., how to properly prepare samples or when to call a detected object an EV. The work described in this thesis aims to support the clinical context by providing technological concepts that could be useful both for fundamental biological studies into the occurrence and behavior of EVs and by giving considerations for sample preparation. It explains why extremely sensitive biosensors are required and describes the development of some lab-on-a-chip solutions.Chapter 1 introduces the concept of liquid biopsy and circulating tumor cells and explains the reasons to explore the feasibility of using EVs instead. It then goes deeper into the composition of EVs and their distribution in the blood. Next, challenges involved in sample preparation are discussed, as well as the barriers to high-throughput reliable detection of EVs.Chapter 2 gives an overview of the Cancer-ID consortium that provided the framework for this (sub)project. It highlights the findings of colleagues in Amsterdam, Delft, Groningen, Twente and Utrecht, who studied EVs from various perspectives in parallel. It gives a comprehensive comparison of the efficacy and throughput of various methods to study EVs for their corresponding applications.Chapter 3 explains the necessity for multi-modal analysis and introduces a first platform that allowed studying individual tumor-derived extracellular vesicles (tdEVs) with scanning electron microscopy, Raman spectrometry and atomic force microscopy to correlate the respectively morphological, biochemical and mechanical data obtained from these single nanoparticles.Chapter 4 reports observations made while performing sample preparation of EVs for different purposes. It was found that a class of polymers, i.e., organosilicon compounds, have a stable interaction with phospholipid membranes found in cells, EVs and artificial membrane models. This has implications for the use of these polymers not only in research but even in daily life, as these silicones occur in cosmetics, pharmaceutics and even food.Chapter 5 presents an integrated system that uses a two-stage identification of tdEVs followed by a two-stage signal amplification. This awarded a highly specific detection method: only tdEVs gave a detectable signal. It also proved to be ultrasensitive; thanks to miniaturization tot the nanoscale of an ELISA-like electrochemical detection mechanism, tdEVs were detectable at concentrations as low as 10 tdEVs/μl.Chapter 6 explores the qualities of a hypothetically ideal biosensor system and provides those solutions that are technologically available to us now.The last chapter discusses how the preceding chapters were able to answer questions existing in the field and what is necessary to further explore the remaining mysteries. |
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