Double loop-stem hairpins mediated hybridization chain reaction: A multifunctional DNA molecular tool to produce the intact aptamer for label-free biosensing

A DNA molecular tool based on the double loop-stem (DL) hairpins mediated hybridization chain reaction (D-HCR) system was proposed to produce the intact G-quadruplex (InG4) aptamers for enzyme-free and label-free biosensing. The D-HCR system was triggered by targets to produce the cross-sequential nanowires of double-stranded and single-stranded DNA (DS nanowire) through the alternating strand displacement events of DL hairpins. Unlike the split G4 (SpG4) aptamers in current HCR, the formation of DS nanowires was accompanied by the release of previously locked InG4 aptamers. Impressively, we tested that the fluorescence enhancement effect of InG4 aptamer on Thioflavin T (ThT) was16.49-fold higher than that of the SpG4 aptamer. To demonstrate the flexibility of the InG4 aptamer-based D-HCR system, we established a fluorescence biosensor depending on the fluorescence enhancement of InG4/ThT with a detection limit of 4.60 pM, and a colorimetric biosensor relying on the catalytic activity of InG4/Hemin with a detection limit of 13.60 pM. The abovementioned biosensors showed high specificity against interfering miRNAs and excellent detection performance in living cells and lysates. Our D-HCR system provides an avenue for preparing DS nanowires to produce the intact aptamer in each hairpin, which may be valuable to prompt the development of HCR-aptamer technique.