To increase the sensitivity of the FID assay, factors such as TO-aptamer ratio and reaction times are very critical. Moreover, the reaction buffer the assay was performed in is also of great importance. The error bars of the results indicate poor reproducibility and that improvements could be done. In this study, the buffer was chosen based on literature of rapid detection of the steroid hormone DHEAS using aptamers. Ebrahimi et al. tested the impact of different concentrations of N aCl and M gCl 2 in 20 µM Tris (pH 8) on the fluorescence of the aptamer with a TO substitution. The authors suggest that HS Tris buffer preserves the secondary structure of the aptamer which facilitates binding of the target. In the current study, the suggestion was translated to the assumption that the HS Tris buffer would facilitate the binding of hydrocortisone to the cortisol aptamer displacing more TO from the aptamer which resulted in a greater decrease in fluorescence. Furthermore, this would correlate with Schaper's theory on the interaction between steroid and nucleotide through a positively charged ionic agent. On the other hand, Sun et al. obtained the most significant fluorescence intensity values of their TO-aptamer complex in ultra-pure water compared to a 50 mM Tris-HCl buffer and PBS buffer. Hence, it is plausible that a different salt concentration in the buffer would improve the sensitivity of this FID assay. Ideally, an experiment analysing the reaction time between the cortisol aptamer and hydrocortisone should have been performed. Instead, the reaction time in this study was based on the results of , who demonstrated a maximum binding of tetracycline to the aptamer after 10 min.