As the mechanism of using TO as a signal molecule upon binding of hydrocortisone to the cortisol aptamer could be utilized, a saturation experiment was conducted to determine the concentration of TO needed to occupy all of the cortisol aptamer's base pairs. Saturation of the aptamer by TO will help to determine the quantity of aptamer to target interactions, and hence, the concentration of cortisol in saliva. It was expected that the fluorescence intensity of TO would increase with increasing TO concentrations. However, a fixed concentration of 0.05 µM cortisol aptamer was used, and hence, it was also expected that the fluorescence intensity would stabilise as the aptamer gets saturated and TO will be exceeding, and thus, not contribute to fluorescence. Figure shows the saturation curve of the cortisol aptamer by TO. The fluorescence intensity increases with increasing concentrations of TO and stabilises around 1 µM TO as the base paired regions of the cortisol aptamer get occupied by TO. Thus, a concentration of 1 µM TO was chosen for further experiments.