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

在开发新药的过程中，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，以便使用可见光或超可见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具有未知的影响，因此需要开发和应用不需要对药物进行任何标记的新方法。本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，这是一种无标记的化学特异性分析技术（该方法可以在没有标记的情况下区分大量化学品），以研究活细胞中的药物量，而无需添加任何标记染料。FTIR光谱法是一种成熟的药物分析方法。然而，它还没有被用于测量活人体细胞中的药物量，因为，首先，它通常不够灵敏，无法检测活细胞中的少量药物。其次，不适合测量活细胞等被大量水包围的样品。幸运的是，这些障碍可以通过使用称为多反射衰减全反射（MB-ATR）的特殊测量模式来克服，该模式可以将灵敏度提高至少10倍，并且允许测量浸泡在水介质中的活细胞。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疾病治疗相关的浓度水平（~10微摩尔）的药物量的可能性。这可以通过使用最佳的反弹次数、ATR材料和测量参数来优化MB-ATR测量来实现，使得信号最大化，同时噪声最小化。然后将这种发展的结果与传统方法的结果进行比较，以证明FTIR技术是研究活细胞中药物的可靠工具。

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.