Toehold-mediated biosensors: Types, mechanisms and biosensing strategies

Toehold-mediated strand displacement (TMSD) reactions between double-stranded DNA and single-stranded oligonucleotides, which use a toehold structure as a switch, enable DNA rehybridization in a fast, base-to-base, programmable and controlled manner. TMSD offers numerous advantages, as reactions are enzyme-free, highly efficient, isothermal and cost-effective. Consequently, TMSD-based reaction systems have attracted great interest from researchers in the last two decades as they become increasingly diverse, versatile and robust, and have been widely used in biochemical sensing and medical diagnosis. In this review, we provide a comprehensive overview of TMSD reactions and their applications in biosensors. The six current TMSD reaction types are generalized according to their various modes of action and nucleic acid conformations. Reaction mechanisms via three major thermodynamic driving forces are explained, along with their dynamic affecting factors, and seven toehold-mediated biosensing strategies are summarized. Furthermore, TMSD-reaction biodetection and bioimaging applications are discussed and, finally, several current challenges and perspectives in the field are considered.