A bivariate fluorescence biosensor based on Janus DNA nanoarchitecture-loaded dual-emissive Ag nanoclusters as bi-responsive signaling reporters

Constructing label-free bivariate fluorescence biosensor would be intriguing and desired for the recognizable and accurate detection of two specific DNA segments, yet the design of functional DNA structures with low overlapped interference might be challenging. Herein in this work, a double-faced J anus D NA n ano a rchitecture ( J DNA) with bi-responsive recognition regions on opposite sides was assembled, which consisted of two substrate strands and two template strands for loading green-/red-emissive Ag nanoclusters ( g AgNC and r AgNC) as bivariate signaling reporters. Of note, the hybridized double helix in the middle rationally oriented two flank faces and stabilized the rigid conformation of J DNA, while the template sequences of bicolor clusters were blocked to minimize non-specific background leakage. Upon inputting two targets, the discernible hairpins lost their hairpin structures due to forming two dsDNA complexes. They were executed to simultaneously invade J DNA for activating two individual target-recycled strand displacement (TRSD) events, guiding signal transduction and efficient amplification. Consequently, the clustering templates were unlocked via the tailored conformation switch of J DNA, in which g AgNC and r AgNC were in situ synthesized in two diagonal positions, thereby significantly emitting bi-responsive signal without cross interference. Benefited from the logic integration of double-faced J DNA and TRSD, a label-free, sensitive and specific bivariate fluorescence approach was developed, which would open a new avenue for the potential application in biosensing and bioanalysis.