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 (<i>bh</i>MB) for improving reaction kinetics and assay sensitivity for a label-free ratiometric fluorescence assay of a specific trigger (<i><b>T</b></i>*), in which red and green silver nanoclusters (<i>r</i>SNC and <i>g</i>SNC) were utilized as two reverse emitting reporters. Unlike traditional dually labeled MB, herein the proposed <i>bh</i>MB was designed with two hairpin subunits for specific recognition and signaling readout. Four functional modules were included: the complement of <i><b>T</b></i>* in the identifiable hairpin, the template sequence of <i>r</i>SNC and <i>g</i>SNC 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 <i><b>T</b></i>*, the conformation switch of <i>bh</i>MB 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 <i>bh</i>MB, the local concentration of reactive species was increased to speed up the reaction kinetics. In a tailored response route, the template sequence for <i>r</i>SNC clustering was merged with increased red fluorescence, while the emission of <i>g</i>SNC 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.