Ratiometric determination of human papillomavirus-16 DNA by using fluorescent DNA-templated silver nanoclusters and hairpin-blocked DNAzyme-assisted cascade amplification.

A signal amplification method has been developed for determination of human papillomavirus-16 DNA (HPV-16 DNA). It consists of a combination of using (a) two-color emitting fluorescent DNA-templated silver nanoclusters (DNA-AgNCs); (b) hairpin-blocked DNAzyme probe (H-Dz), and (c) catalytic hairpin assembly. In the absence of the model target HPV-16 DNA, the H-Dz forms a hairpin structure through intramolecular hybridization. It inhibits the catalytic activity of the DNAzyme. However, in the presence of HPV-16 DNA, it will hybridize with the DNAzyme probe which leads to the opening of the hairpin and the formation of an active secondary structure in the catalytic core. This produces an "active" DNAzyme which can cut H-Dz under the catalytic action of Mg(II) ions. Then, the DNA fragment and HPV-16 DNA are separated due to the weak hybridization. The former triggers a downstream catalytic hairpin assembly (CHA). This changes the color of the fluorescence of DNA-AgNCs from red (630 nm) to yellow (570 nm). The HPV-16 DNA will hybridize with another H-Dz to trigger the next round of activation cycle to cut H-Dz. In this way, recycling amplification results in the generation of a strong signal that allows human HPV-16 DNA to be detected with a 5.7 pM detection limit. Graphical abstract A simple and effective cascade signal amplification method was developed for fluorometric determination of HPV-16 DNA. It is based on a combination of chameleon fluorescent DNA-AgNC, hairpin-blocked DNAzyme probe and catalytic hairpin assembly (CHA). Target recycling amplification results in significant signal changes, thus providing high detection sensitivity.