A Ready-to-Use Recombinant Yeast Two-Hybrid Assay for Thyroxine Detection

We report a freeze-dried ready-to-use yeast thyroid screen (YTS), preserving the general dose–response characteristics of the freshly prepared counterpart. This field-deployable method reduces the assay time of the overall procedure from several days to 5 h with no requirement for sterile conditions, thus fulfilling key requirements for on-site implementation in a biosensor array. 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). Long-term stabilization is demonstrated using spiked (T4, 2 µM) river water and extracted wastewater effluent. After 5 months of storage, the T4-equivalent activities were 96 ± 38% and 112 ± 15% for river water and wastewater, respectively. In summary, we have successfully demonstrated a proof of principle of a field-deployable yeast thyroid screen (YTS) by using freeze-dried cells and trehalose as a cryoprotectant to achieve storability for up to 5 months at 4 °C.