Sensitive Microscale Thermophoresis Assay Using Aptamer Thermal Switch

Rapid sensitive detection of targets is desired in broad fields, such as environmental monitoring, food safety, and molecular diagnosis. We demonstrated an innovative strategy of constructing aptamer thermal switch for target detection, which affords a large difference in heating-induced conformation changes between the bound and the unbound states. This aptamer thermal switch is obtained by rational engineering aptamer sequence and modulating aptamer conformation stability under heating. We further introduced a conformation-sensitive fluorophore into the aptamers, converting heating-induced conformation changes to heating-induced fluorescence changes. The bound and unbound aptamers showed a large difference in heating-induced fluorescence change. Using these aptamers in microscale thermophoresis (MST), which is a powerful tool for quickly measuring fluorescence responses to heating with precise ratiometric analysis, we achieved detection of a variety of targets (e.g., cadmium ions (Cd²⁺), small molecule aflatoxin B1, and protein biomarker immunoglobulin E) within seconds with pronounced sensitivity. We envision that this strategy will have wide applications to detection of targets of interest in broad research fields, and it will also be helpful for affinity binding study.