Homogeneous and Universal Detection of Various Targets with a Dual-Step Transduced Toehold Switch Sensor

Toehold switch sensors represent a class of new advances that allow specific targets to trigger in situ expression of a protein reporter. Although they offer unique advantages of a label-free nature and high portability, they generally require repeated sequence design, high expenditure, and laborious optimization of toehold switch sequences according to different targets. To simplify the sensing process further and to improve its practicability, we innovatively introduce a dual-step pre-transduction upon traditional toehold switch sensor. Through two successive toehold-mediated strand-displacement reactions that are initiated, respectively, by a linear and an associative trigger, different DNAs, RNAs, or ligands of functional nucleic acids can be generally transduced into the input of one high-performance toehold switch sensor. This advance greatly increases the target range. Furthermore, the whole process is signal-on, homogeneous, and free of any requirements for complicated operations such as probe labeling, separation, and reconstruction of the toehold switch, being promising and practical even in portable or point-of-care assays.