Development of a nanoscale, near-infrared spectroscopy imaging tool for in situ, rapid and label-free analysis of single extracellular vesicles

开发纳米级近红外光谱成像工具，用于单个细胞外囊泡的原位、快速、无标记分析

• 批准号: BB/X004449/1
• 负责人: Wayne Nishio Ayre
• 金额: $ 23.83万
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX004449%2F1
• 关键词: Development; nanoscale; near; infrared; spectroscopy

Cells release small spheres, known as extracellular vesicles (EVs), which are approximately 1000 times smaller than the width of a human hair (nanoscale). Recent research has shown that these EVs contain a cargo of signalling molecules that can act to either maintain health (e.g. blood vessel formation, immune response) or encourage disease progression (e.g. cancer, Parkinson's disease). As a result, the biological role and therapeutic potential of EVs has gained significant interest. Moreover, the discovery that EVs are present in circulating blood and elevated in certain diseases has also increased their potential use in diagnostics. A major limitation to the evolution of this field of research however has been the limited techniques available to easily analyse EVs. Many of the research techniques to study EVs require specialist equipment and training as well as significant time, sample processing and labelling, which may induce artefacts or bias results. Due to the low abundance of EVs and their contents, many existing techniques also pool thousands to millions of EVs for single analysis, assuming a homogenous population, when in fact studies have shown from a single cell type, several different sub-populations of EVs are present. To truly understand the biological role of EVs in health and disease and their therapeutic and diagnostic potential, a closer look at the heterogeneity of single EVs is needed in a manner that is both high-throughput and label-free whilst simultaneously maintaining EVs in their natural state. IR spectroscopy may offer a solution to this problem. It is based on the fact that molecules vibrate at specific frequencies due to the stretching and bending of the chemical bonds between atoms. When a chemical bond is exposed to an infrared light at the same frequency as it vibrates, the bond will absorb the energy. Although this technique has provided scientists with the ability to study chemical bonds in great detail, only recently has the technology advanced to the point where it can be applied to samples at a nanoscale. This cutting-edge approach, known as photo-induced force microscopy (PiFM), uses an extremely fine tip to measure the vibrational energy of molecules whilst they are excited by infrared light. Such an approach allows the chemical composition and topography of dry samples to be characterised at a nanoscale. However infrared light is highly prone to absorption in water and the movement of EVs due to their surface charge (Brownian motion) would make locating EVs in liquids using PiFM extremely challenging as well as prone to high levels of background noise. These technical challenges will be overcome in this project using two novel approaches. Firstly, IR light with a shorter wavelength, known as near-IR, will be used as it is less affected by water. Secondly, devices with unique surface features and different materials will be manufactured that can amplify the near-IR signal in water. These surfaces will also be chemically functionalised to capture the EVs for easy localisation and analysis with the PiFM. Analysing near-IR absorption is a complex task as this region of the IR spectrum consists of signals arising from combinations of chemical group vibrations as well as overtones (multiples of chemical vibrations). The project will therefore require a simplified model of EVs (empty vesicles composed of lipids, also known as liposomes) and advanced computational techniques (i.e. machine learning) to develop a database of near-IR chemical signals. Once the technology is optimised and refined, it will be validated and tested using cell derived EVs. This project will therefore develop a label-free, non-invasive, rapid technology to analyse the size and chemical composition of EVs in their natural state at a single vesicle level, providing information on the heterogeneity of EV populations and helping discover potential future therapeutic and diagnostic markers.

细胞释放小球，称为细胞外囊泡（EV），比人类头发的宽度（纳米级）小约1000倍。最近的研究表明，这些EV含有信号分子的货物，可以起到维持健康（例如血管形成，免疫反应）或促进疾病进展（例如癌症，帕金森病）的作用。因此，EV的生物学作用和治疗潜力获得了极大的兴趣。此外，EV存在于循环血液中并在某些疾病中升高的发现也增加了它们在诊断中的潜在用途。然而，这一研究领域发展的一个主要限制是可用于轻松分析电动汽车的技术有限。研究电动汽车的许多研究技术需要专门的设备和培训，以及大量的时间，样品处理和标签，这可能会导致伪影或偏倚结果。由于EV及其含量的低丰度，许多现有技术还汇集了数千至数百万EV用于单一分析，假设是同质群体，而实际上研究已经表明，从单一细胞类型，存在几种不同的EV亚群。为了真正了解EV在健康和疾病中的生物学作用及其治疗和诊断潜力，需要以高通量和无标记的方式仔细研究单个EV的异质性，同时保持EV处于自然状态。红外光谱法可以解决这个问题。它基于这样一个事实，即由于原子之间的化学键的拉伸和弯曲，分子以特定的频率振动。当一个化学键暴露在与其振动频率相同的红外光下时，该键将吸收能量。虽然这项技术为科学家提供了详细研究化学键的能力，但直到最近，这项技术才发展到可以应用于纳米尺度的样品。这种尖端的方法被称为光诱导力显微镜（PiFM），使用极细的尖端来测量分子的振动能量，同时它们被红外光激发。这种方法允许干燥样品的化学成分和形貌在纳米级上进行表征。然而，红外光在水中非常容易被吸收，并且由于电动汽车的表面电荷（布朗运动），电动汽车的运动将使得使用PiFM在液体中定位电动汽车极具挑战性，并且容易受到高水平的背景噪声的影响。这些技术挑战将在本项目中使用两种新的方法来克服。首先，将使用波长较短的红外光（称为近红外光），因为它受水的影响较小。其次，将制造具有独特表面特征和不同材料的设备，可以放大水中的近红外信号。这些表面还将进行化学功能化，以捕获EV，从而便于使用PiFM进行定位和分析。分析近红外吸收是一项复杂的任务，因为红外光谱的这个区域由化学基团振动以及泛音（化学振动的倍数）的组合产生的信号组成。因此，该项目将需要一个简化的EV模型（由脂质组成的空囊泡，也称为脂质体）和先进的计算技术（即机器学习）来开发近红外化学信号数据库。一旦该技术得到优化和完善，它将使用细胞衍生的电动汽车进行验证和测试。因此，该项目将开发一种无标记，非侵入性，快速的技术，以在单个囊泡水平上分析EV在自然状态下的大小和化学组成，提供EV群体异质性的信息，并帮助发现潜在的未来治疗和诊断标志物。