As observed from the results, TO is a good indicator for interaction between the cortisol aptamer and the target, hydrocortisone, in HS Tris buffer, as the decrease in fluorescence is caused by the aptamer-target interaction and not the interaction between hydrocortisone and TO. A similar observation was made by Sun et al. using a tetracyclinebinding aptamer and TO to determine concentrations of tetracycline in raw milk. The study gained a high sensitivity using the mechanism with a LOD in the ng range of 19 ng/L, proving the great potential of using a label-free fluorescent aptasensor. In comparison to labeled fluorescent aptasensors, which requires modification of the aptamer for detection of biomolecules, label-free fluorescent aptasensor is less time-consuming, labour intensive, and cheaper to produce. Furthermore, the label-free aptasensor prevents structural or conformational changes in the aptamer and limits the risk of affecting the aptamer-target affinity and selectivity . However, the LOD of this study was previously determined to be around 0.2 µM hydrocortisone, indicating that optimisation is needed to determine the low concentration of cortisol in saliva. Another way of increasing the signal of the assay could be to change the fluorescent dye. Furthermore, the use of label-free aptasensors is an advantage compared to more traditional analysis methods such as ELISA and HPLC. HPLC requires large and expensive instruments and trained personnel which centralises the analysis. It makes the analysis limited and difficult to perform on-site, and thus, hinders a rapid and real-time application of the method. ELISA is a very sensitive analysis methods and can measure concentrations in the µg/L or lower range. However, the method is also costly and can show poor selectivity for a desired target if similar molecules with minor differences in the functional groups are present . Steroid hormones have a similar backbone, and high selectivity is therefore a requirement to distinguish between them to perform a precise analysis using this technique. Aptamers have shown to be selective against different steroid hormones. This selectivity was demonstrated by who analysed the aptamer's selectivity against other steroid hormones. For other small molecules aptamers have shown high selectivity against minor structural derivations. For example, demonstrated high selectivity of aptamers by comparison of the binding between the tetracycline-aptamer and tetracycline against similar structures to the tetracycline such as oxytetracycline. This proves the great potential of using aptamers for determination of biomarker in complex matrices such as saliva in the future.