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A rapid signal amplified DNA detection method based on self-replicating catalyzed hairpin assembly (SRCHA) has been proposed. In this SRCHA system, two split target DNA sequences were respectively integrated into hairpin auxiliary probes H1 and H2. H2 was used as fluorescent probe which containing a fluorescent nucleotide base analog pyrrolo-deoxycytidine (P-dC) at the end of the stem. Target DNA can be circularly used in this SRCHA system to form the helix DNA H1-H2 complex, the structure change of H2 will move P-dC from hairpin stem to flexible ssDNA sticky end, leading to fluorescence increase due to the less stacking interaction. Meanwhile, the two spilt target DNA sequence was reunited and the target DNA replicate was obtained, which also can be circularly used as new activator to trigger additional CHA reaction and fluorescence signal was then rapidly and significantly enhanced. This SRCHA system has been successfully employed for DNA detection with picomolar within around 15min, and provides a potential technology for the real-time rapid bioanalysis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In the present work, an enzyme-free and signal amplified nucleic acids detection method based on the fluorescent nucleotide base analog pyrrolo-deoxycytidine (P-dC) and catalyzed hairpin assembly (CHA) has been developed. In the CHA signal amplification system, two hairpin auxiliary probes, H1 and H2, which containing a fluorescent P-dC at the end of the stem, respectively, were used as the fluorescent probes. The fluorescence of P-dC in H1 and H2 was quenched owing to the stacking interaction among the bases in the stem. In the presence of the target DNA, the catalytic assembly of H1 and H2 was triggered and the target could be released during the helix DNA H1-H2 complex formation process, then the released target was used to trigger another reaction cycle. In the H1-H2 complex, P-dC was located in the flexible single-stranded DNA (ssDNA) sticky ends instead of the rigid stem, thus resulting in the increase of fluorescence. The cycling use of the target in the CHA system amplified the fluorescence signal, and the detection limit of this method was obtained as 19pM, which is 3 orders of magnitude sensitive than the conventional fluorescent nucleotide base analogs based approach without CHA signal amplification., (Copyright © 2016 Elsevier B.V. All rights reserved.)