A User Ready Recombinant Yeast Two-Hybrid Thyroxin Assay

We report a freeze-dried ready-to-use yeast thyroid screen (YTS), displaying detection sensitivity and performance comparable to that of the freshly prepared counterpart. This field-deployable method reduces the duration of the worktime from several days to 5 hours with no requirement for sterile conditions, fulfilling key requirements for implementation in a biosensor array with on-site application. The effects of cell density and concentration of the cryoprotectant trehalose on median effective concentrations (EC₅₀), limit of detection (LOD) and biosensor induction (IF) were determined and monitored over a storage period of 5 months. In addition, the impact of these parameters was monitored on the biosensor survival rate during freeze-drying and the subsequent storage process. Throughout the 5-month study, the freeze-dried recombinant yeast-assay retained comparable dose-response characteristics to those of the freshly prepared counterpart, displaying median values of EC₅₀ in the range of 350 nM to 550 nM and LODs in the range of 20 nM to 45 nM of the reference compound thyroxine (T4). A proof of principle of the long-term stabilization is demonstrated using spiked (T4, 2 µM) river water and extracted wastewater effluent. After 5 months of storage, the T4 recoveries were 96 ± 38% and 112 ± 15% for river water and wastewater, respectively.