Target-Catalyzed Assembly of Pyrene-Labeled Hairpins for Exponentially Amplified Biosensing

Rapid and sensitive detection of nucleic acids is vital for disease diagnosis. This work designed an enzyme-free isothermal strategy for rapid exponential signal amplification through target-triggered catalytic hairpin assembly (CHA) to induce the spatially sensitive fluorescent signal of the pyrene excimer. Functionally, this system consisted of three pyrene labelled hairpins (H1, H2, and H3) and one catalyst DNA C. In the presence of C, the CHA was activated to generate intermediate I, which contained a single-stranded region identical to the C sequence for initiating the second cycle of CHA to obtain 2I and thus achieved the exponential formation of I along with the switching of pyrene excimer. The fluorescent signal of the pyrene excimer could be further enhanced via the inclusion of γ-cyclodextrin and showed a linear increase upon increasing logarithm of C concentration. Through the introduction of a helping hairpin H4-containing C sequence and a region specific to the target, this strategy could be extended to realize the quick and sensitive detection of different analytes. Using dengue virus RNA as an analyte model, the proposed fluorescent method showed a linear range from 0.1 to 50 nM with a limit of detection of 0.048 nM at 3σ and good selectivity. The excellent performance and convenient operation demonstrated its promising application in clinical disease diagnosis.