A simplified antibody-responsive fluorescence biosensor by proximity ligation-induced quasi-Y-DNA assembly to illuminate emissive DNA looped-Ag nanocluster

Exploring the proximity ligation-induced quasi-Y-DNA assembly (plqYDA) for creating label-free fluorescence biosensor might be intriguing based on DNA loop-hosted red Ag nanocluster (lrAgNC) as a signaling reporter. For proof-of-concept, a bivalent digoxigenin antibody (anti-Dig) with two identical binding sites was used as an analyte model, and it can specifically distinguish two Dig haptens that were separately modified in two modular split strands (SS1 and SS2) with complements each other and invading domains. The strong and robust anti-Dig-Dig binding guided the proximity ligation of SS1 and SS2 to construct geometric-structure plqYDA with a paired stem and two sticky linkers. After introducing a dsDNA duplex from a reporting strand (RS) with seven-polycytosine template (C₇) of lrAgNC and a blocking strand (BS), entropy-driven hybridization and cooperative displacement occurred. As a result, RS was rationally immobilized in plqYDA to crimp single-coil C₇ as a closed and extruded loop, which preferably favored the development of emissive lrAgNC when adding AgNO₃ and NaBH₄. By collecting the distinct fluorescence signal, the label- and enzyme-free assay of anti-Dig was readily achieved, exhibiting good specificity and high sensitivity. This would be the first example of our best understanding of integrating plqYDA and lrAgNC for applicable biosensing and bioanalysis.