The saturation of an aptamer was determined by the highest % increase in fluorescence when the fluorescence intensity of different concentrations of TO is compared to the fluorescence intensity of TO with added aptamer (eq. 1). The saturation ratio of TO and the aptamer was calculated with a fixed concentration of TO and varying aptamer concentrations. All experiments were performed in a high salt (HS) Tris buffer. Prior to the saturation experiments of cortisol, a standard curve of TO was prepared where solutions of 150 µL were prepared. TO solutions of increasing concentrations (0, 0.005, 0.05, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5 µM) in HS Tris buffer were prepared in a 96-well black microplate (Greiner Bio-1 96 or 384 well microplate) (Kremsmünster, Austria). Solutions were vortexed in the plate for 1 min prior to addition. Fluorescence was measured with a BMG Labtech Fluostar Omega Microplate Reader (Offenburg, DE) at and excitation wavelength of 485 nm, emission wavelength of 544 nm, a gain value 2000, with 10 seconds of shaking. To determine the saturation of aptamer, and thereby determine the optimal ratio of TO:Aptamer, 1.5 µL of 5 µM aptamer (final concentration 0.05 µM) was added to each of the TO solutions. The fluorescence was measured at the same settings as mentioned above. To establish the optimal ratio of TO/Aptamer, the concentration of TO was fixed to 0.5 µM while the concentrations of aptamer varied (0, 0.1, 0.5, 2.5, 5, 15, 25, 50 µM). A 5 µL aliquot of each aptamer solution was prepared in TE buffer. Aliquots of 150 µL 0,5 µM TO solution was prepared in each in the 96-well black microplate (Greiner Bio-1 96 well microplate) (Kremsmünster, Austria). The plate was vortexed for 1 min. Fluorescence intensity of TO solutions was measured at excitation 485 nm, emission 544 nm, gain value 2000, and 10 sec. shaking. 1.5 µL of aptamer solutions in increasing order of concentration (final concentrations 0.001, 0.005, 0.025, 0.05, 0.15, 0.25, 0.5 µM) were added to each well of 0.5 µM TO solution respectively. Plate was vortexed for 1 min. Fluorescence was measured as mentioned above. All assays were performed at room temperature.