Introduction and summary:
Extracellular vesicles (EVs) are tiny particles released from biological cells (diameter approx. 100 nm). EVs can circulate in the body, while cells are mostly fixed in tissue. EVs are considered a mechanism for communications between cells, allowing cells to exchange proteins, lipids and genetic material. Elevated levels of EVs have been associated with several disease states such as atherosclerosis, diabetes, cancer, arterial cardiovascular diseases and venous thromboembolism. EVs thus contain information about the cells in the body. This information can be revealed by characterising the EVs in a drop of blood. New methods to characterise and analyse EVs are essential to understand the biological functions of these vesicles, and to develop new clinical methods involving their use and/or analysis.
The aim of the project is to develop high throughput chemical analysis of EVs and facilitate medical research on EVs using the proposed new system. An optical chip will be developed to characterise the EVs using Raman-spectroscopy. One or a few EVs will be held by optical forces for long enough to analyse them. This will be done at several sites on the chip, using light from a single laser. The chip will be connected with a bundle of optical fibres to an optical spectrometer, that will acquire the Raman-spectra from the EVs. Based on the Raman-spectra, chemical information about the EVs can be obtained. The system will allow many EVs to be analysed at the same time, and thus make it a high throughput system.
Status per November 2023
The main hypothesis of the project is that a trench in a waveguide can be used for trapping and Raman spectroscopy of EVs. It has now been experimentally demonstrated that the chosen waveguides are suitable, as their Raman background is low. It has also been shown that analysis with machine learning works very well on Raman spectra from collaboration partners. The first result has been published and the second submitted. But it has not been possible to obtain satisfactory quality for the trench across the waveguide and the chip thus does not work yet for trapping and Raman spectroscopy. This is because the material, silica, is difficult to etch, as well as the replacement of personnel, as mentioned in 2022. The work on etching the trench will continue with a new procedure. The goal is to make it work in the first half of 2024. The waveguides, the experimental setup and the analysis method thus work, but the trench is an important missing feature to achieve the desired result. Efforts will be made to make measurements in parallel, to reach the aim of high throughput.
Rabiul Hasan defended his thesis on 14th September 2023 and Mathias N. Jensen defends 7th Dec. 2023. Thus, the project has graduated two PhDs, as planned. Rabiul Hasan has returned to Bangladesh, to a position as associate professor. He will continue to collaborate with the project, with the aim of writing two articles in 2024. Mathias N. Jensen will continue as a postdoc on the project for one year, followed by two years financed with internal funds. This is a very good solution for the project, as it was not possible to find someone for this position in 2022/2023. In addition, the project has financed Jehona Salaj for four months as a researcher. She is doing a PhD on another project and is helping on this project with the production of the chip. She will thus be very central in getting the trench and the chip to work in 2024.
The number of published articles from the project has increased in 2023 and is now on track. But there have been few conference contributions, partly due to corona. In 2024, emphasis will be put on obtaining many publishable results and participation in conferences.
Extracellular vesicles (EVs) are bilayer membrane vesicles released from various cells into their surroundings. EVs include exosomes (30-100 nm in diameter) and microvesicles (100-1000 nm) and express surface antigens specific of parental cells. EVs are considered a mechanism for intercellular communications, allowing cells to exchange proteins, lipids and genetic material. Elevated plasma levels of EVs have been associated with several disease states such as atherosclerosis, diabetes, cancer, arterial cardiovascular diseases and venous thromboembolism. New methods to characterise and analyse EVs are essential to understand the physiological and pathological functions of these vesicles, and to develop new clinical methods involving their use and/or analysis.
The aim of the project is to develop high throughput chemical analysis of EVs and facilitate medical research on EVs using the system developed. Dielectric optical waveguides will be tapered down to sub-micron tips. A tightly confined and standing wave between two tips will be used for optical trapping and Raman-spectroscopy of EVs. This will be done at several sites on the chip simultaneously. As a very small amount of the incident light is ‘consumed’ by each particle, the incident light can be re-used at several sites in series, without increasing the input power. This is, to the best of our knowledge, a completely new concept. By also doing this in parallel, a 2D array of trapping sites can be made, but this time by multiplying the input power. The Raman-scattered light will be collected by waveguides with high numerical aperture and directed to a 1D fibre array, which connects the chip with a multichannel spectrometer.
Two research groups in medical biology participate in the project and will use the system developed for research on venous thromboembolism and novel antimicrobial molecules.