Target-Responsive Conformation Switch of Bihairpin Molecular Beacon to Illustrate Ratiometric Fluorescence Biosensing of Dual-Emissive Silver Nanoclusters

It might be intriguing to explore the tailored ability of a bihairpin-structured molecular beacon (bhMB) for improving reaction kinetics and assay sensitivity for a label-free ratiometric fluorescence assay of a specific trigger (T*), in which red and green silver nanoclusters (rSNC and gSNC) were utilized as two reverse emitting reporters. Unlike traditional dually labeled MB, herein the proposed bhMB was designed with two hairpin subunits for specific recognition and signaling readout. Four functional modules were included: the complement of T* in the identifiable hairpin, the template sequence of rSNC and gSNC in the signaling hairpin, two variable consensus loops capable of complementarily hybridizing to form one paired stem, and two stems able to merge into one new loop. Upon introducing T*, the conformation switch of bhMB was triggered via affinity linking to a cognate consensus sequence, thereby reconstructing a perfect stem-loop monohairpin structure. As such, the mutual transition of loops and stems between nonbinding and binding states was powered by a population-shift mechanism to shift the thermodynamic equilibrium toward the binding state. Benefiting from the structure confinement of bhMB, the local concentration of reactive species was increased to speed up the reaction kinetics. In a tailored response route, the template sequence for rSNC clustering was merged with increased red fluorescence, while the emission of gSNC was decreased, pointing to the obtained specific and sensitive ratiometric signal via built-in correction. Thus, this label-free strategy would be the first example to integrate recognizing and signaling subunits in a double-hairpin beacon for label-free biosensing and potential applications.