Label-Free in Situ Quantification of Drug in Living Cells at Micromolar Levels Using Infrared Spectroscopy

Quantifying the rate and the amount of drug entering live cells is an essential part of the medicine development process. Infrared spectroscopy is a label-free, chemically selective tool for analyzing the composition of live cells in culture that has the potential to quantify, in situ, the amount of drug entering living cells in a nondestructive manner, although its sensitivity is currently limited. This paper is the first to demonstrate in situ quantification of the cancer drug, fluorouracil, in live cells at a therapeutically relevant concentration using Fourier transform infrared spectroscopy. To achieve the required improvement in detection and quantitation limits of the IR measurement, two strategies were exploited. First, a sampling method called multibounce attenuated total reflection was used to optimize the signal while second, a long pass filter in combination with a mercury cadmium telluride detector was used to reduce the instrument noise. Using these novel adaptations, it was possible to quantify 20 mu M of fluorouracil in cell culture medium using a standard FTIR instrument, while it was possible to quantify and measure the flux of fluorouracil in situ in living cells treated with an 80 mu M drug.