Quantification of drug in live cells using label-free multi-bounce attenuated total reflection Fourier Transform Infrared (MB-ATR FTIR) spectroscopy

在开发一种新药的过程中，了解人体细胞吸收了多少药物是很重要的。这通常是通过添加染料来标记药物来实现的，以便使用可见光或超紫外光来可视化吸收到细胞中的药物量。然而，添加的标记染料可能对总体结果有未知的影响，因此有必要开发和应用一种不需要任何药物标记的新方法。该项目将研究如何使用一种称为傅里叶变换红外（FTIR）光谱的方法，这是一种无标记而化学特异性的分析技术（这种方法可以在没有标记的情况下区分大范围的化学物质），来研究活细胞中药物的数量，而无需添加任何标记染料。FTIR光谱是一种成熟的药物分析方法。然而，它还没有被用于测量活细胞中的药物量，因为，首先，它通常不够灵敏，无法检测到活细胞中的少量药物。其次，不适合测量被大量水包围的样品，如活细胞。幸运的是，这些障碍可以通过使用一种特殊的测量模式来克服，这种模式被称为多反弹衰减全反射（MB-ATR），它可以将灵敏度提高至少10倍，并且允许测量在水介质中浸泡的活细胞。该项目将使用一种治疗癌症的药物（阿霉素），首次证明使用FTIR技术测量活细胞中与治疗疾病相关的药物浓度水平（~10微摩尔）的可能性。这可以通过优化MB-ATR测量来实现，通过使用最佳的弹跳次数、ATR材料和测量参数，使信号最大化，同时使噪声最小化。然后将这一发展的结果与传统方法的结果进行比较，以证明FTIR技术是研究活细胞药物的可靠工具。

In the process of developing a new medicine, it is important to know how much of the medicine is absorbed by a human cell. This is usually achieved by adding a dye to label the medicine in order to visualise the amount of the medicine absorbed into the cell using visible or ultra-valet light. However, the added labelling dye may have an unknown effect to the overall results, therefore there is a need to develop and apply a new method that does not require any labelling of the medicine. This project will investigate on how to use a method called Fourier Transform Infrared (FTIR) spectroscopy, which is a label-free while chemically specific (this method can distinguish a large range of chemicals without labelling) analytical technique, to study the amount of medicine in living cell without any added labelling dye. FTIR spectroscopy is a well-established method for the analysis of medicine. However, it has not been used for measuring the amount of medicine in living human cell because, first, it is normally not sensitive enough to detect the small amount of medicine in a living cell. Second, it is not suitable to measure samples that are surrounded by a large amount of water such as live cells. These obstacles, fortunately, can be overcome by using a special measurement mode called the multi-bounce attenuated total reflection (MB-ATR) which can increase the sensitivity by at least 10 times as well as allowing the measurement of live cells bathing in aqueous medium. The project will use a medicine for treatment of cancer (doxorubicin) to demonstrate, for the first time, the possibility of using the FTIR technique to measure the amount of the medicine in living cells at concentration levels that is relevant to the treatment of the disease (~10 micro molar). This can be achieved by optimising the MB-ATR measurements through using the optimal number of bounces, ATR materials and measurement parameters so that the signal is maximised while the noise is minimised. The result of this development will then be compared to the results from the traditional methods to demonstrate that the FTIR technique is a reliable tool for studying medicine in living cell.