Cavity enhanced absorption spectroscopy for drug quantification in living cells

用于活细胞药物定量的腔增强吸收光谱

• 批准号: 2241890
• 金额: --
• 依托单位: UNIVERSITY OF EXETER
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2241890
• 关键词: Cavity; enhanced; absorption; spectroscopy; drug

Aim: The aim of this project is to develop a generic label free spectroscopic method, based on cavity enhanced absorption spectroscopy, for quantifying drug intake in living cells: e.g. chemotherapy drugs entering cancer cells.Historically drugs entering cells have been quantified by lysing the cell and measuring drug levels in the cell lysate, for example by fluorescence spectroscopy or mass spectrometry. Although this method provides important insights into the problem of drug permeation it is restricted to cell population analysis. Recently Raman Spectroscopy has been used as a detection tool to probe drug permeation directly inside cells but although promising, Raman Spectroscopy currently remains semi quantitative with the Raman signal being dependent on the complex cellular environment. During this PhD studentship a novel Cavity-Reflection-Enhanced Light-Absorption spectrometer optimized for single cell absorption measurements will be developed. This is a miniaturized single cell spectrometer with a miniaturized detection chamber down to the cell volume and optimized for high sensitivity UV-Vis absorption measurements in cells. This platform will allow to investigate and optimise efficacy of drug intake in cells.Workload Milestones: 1) Technology development. The PhD student taking part in this project will be involved in building and optimizing the optical setup, including fibre alignment, fibre micromachining and generating semi-transparent aluminium mirrors. Methods for separating optical signals from the drug, the cell, and the background will be developed by referencing measurements at different wavelengths ( e.g. tryptophan absorption peak at 290 nm). 2) Chemotherapy Resistance. To validate the technology for cancer research we will start by investigating the drug, doxorubicin, which enters a human lung cancerous cell-line. In this purpose COR-L23/R, a multi-drug resistant human lung cancer cell-line over expressing efflux pumps that export doxorubicin out of the cell will be compared to its parent line COR-L23. By analysing the spectral difference at the single cell level we will provide a platform to rapidly investigate the ability of doxorubicin, and other chemotherapy drugs, to permeate through the cellular membrane of cancer cells. The kinetics of cellular uptake and kinetics of resultant cell response will be investigated on this platform in multiplexed single-cell measurements. The effects of cell heterogeneity on drug efficacy will be quantified; variations of the molecular structure of a drug can will be tested in order to optimise dose and response.

目的：本项目的目的是开发一种基于腔增强吸收光谱的通用无标记光谱方法，用于定量活细胞中的药物摄取：例如进入癌细胞的化疗药物。历史上，进入细胞的药物是通过裂解细胞和测量细胞裂解产物中的药物浓度来定量的，例如通过荧光光谱或质谱学。虽然这种方法对药物渗透问题提供了重要的见解，但它仅限于细胞群体分析。最近，拉曼光谱已被用作直接探测药物在细胞内渗透的检测工具，但尽管前景看好，拉曼光谱目前仍然是半定量的，拉曼信号依赖于复杂的细胞环境。在此博士研究生期间，将开发一种新的腔反射增强光吸收光谱仪，该光谱仪优化了单细胞吸收测量。这是一种小型化的单细胞光谱仪，带有一个小型化的检测室，精确到细胞体积，并针对细胞内高灵敏度的UV-Vis吸收测量进行了优化。该平台将允许调查和优化细胞内药物摄取的效果。工作量里程碑：1)技术开发。参与该项目的博士生将参与构建和优化光学设置，包括光纤对准、光纤微加工和生成半透明铝镜。将通过参考不同波长的测量(例如，290 nm处的色氨酸吸收峰)来开发从药物、细胞和背景中分离光信号的方法。2)化疗耐药。为了验证癌症研究的技术，我们将从研究药物阿霉素开始，阿霉素进入人类肺癌细胞系。为此，将多药耐药人肺癌细胞系COR-L23/R与其亲本株COR-L23进行比较。通过在单细胞水平上分析光谱差异，我们将提供一个平台来快速研究阿霉素和其他化疗药物通过癌细胞细胞膜的能力。细胞摄取的动力学和由此产生的细胞反应的动力学将在该平台上以多路单细胞测量进行研究。将量化细胞异质性对药物疗效的影响；将测试药物罐分子结构的变化，以优化剂量和反应。