Hairpin Hybridization Assembly to Catenate Ag Nanocluster-Riveted Hoogsteen Triplexes for Amplified Fluorescence Biosensing

The inherent ability of hairpin hybridization assemblies (HHA) to catenate Ag nanocluster (<i>c</i>AgNC)-riveted Hoogsteen triplexes was explored to present a label-free fluorescent biosensing response to a specific external initiator (<b><i>I</i></b>*). Among the four functional hairpin reactants needed for operating HHA, a pH-responsive triplet motif of mirror-repeated homopurine-homopyrimidine is grafted on the end of two species, preferentially adopting an intramolecular triplex conformation under slightly acidic conditions and then forming a dimer under the riveting of a <i>c</i>AgNC synthesized in the seven polycytosine bases (C₇) closed loop of the triplexes. Upon introduction of <b><i>I</i></b>*, specific recognition and affinity binding occurred to execute progressive HHA events via sequential unfolding and cross-base-pairing. In the resultant periodic nicks in the as-generated long linear product, numerous repeats of two triplex stretches were located to orient each pair of two C₇ splits closely. Thus, <i>c</i>AgNC concatemers as stabilizing nanorivets are developed to emit cooperatively enhanced red fluorescence, enabling sensitive detection of targeted <b><i>I</i></b>* in a tailored reaction route. Predictably, <i>c</i>AgNC and triplex dimers cannot be formed in the absence of <b><i>I</i></b>* due to the remote triplex stretches in the intact hairpin reactants. Compared with non-HHAs, our HHA-cascading <i>c</i>AgNC emitters achieved 13-fold signal amplification for detecting <b><i>I</i></b>* at an ultralow level. This label-free fluorescence strategy would suggest the first example of advancing simple assay platforms for biosensing and bioanalysis applications.