Detection Of DNA By HRP - Mimicking DNAzyme Catalytic Chemiluminescence System Based On Exonuclease Ⅲ Assisted Signal Amplification

The ability to detect and sense ultralow concentrations of specific DNA sequences by using simple and low-cost assays is important in clinical diagnostics, mutation detection, and biodefense applications. Therefore, the fabrication of label-free and high sensitivity of DNA detection biosensors has become highly focused in analytical chemistry. We established an amplified chemiluminescence (CL) biosensing platform for ultrasensitive DNA detection. It is based on the Exonuclease III (Exo III)-assisted target recycling amplification and catalytic effect of G-quadruplex-hemin DNAzyme to stimulate the generation of CL in the presence of H2O2and luminol.1. G-Quadruplex DNAzyme-based chemiluminescence biosensing strategy for ultrasensitive DNA detection:combination of Exonuclease Ⅲ-assisted signal amplification and carbon nanotubes-assisted background reducingWe report an amplified CL biosensing platform for ultrasensitive DNA detection based on the Exo Ⅲ-assisted target recycling amplification and catalytic effect of G-quadruplex-hemin DNAzyme to stimulate the generation of CL in the presence of H2O2and luminol. Moreover, the typical problem of high background induced by excess hemin itself can be effectively addressed through the absorbing of superfluous hemin on the surface of single-walled carbon nanotubes and then removing though centrifugation. Therefore, our proposed biosensing exhibited a high sensitivity toward target DNA with a detection limit of12fM, which was about100-fold lower than that of the DNAzyme-based CL sensor for DNA detection without Exo Ⅲ-assisted amplification. This sensing platform provides a label-free and cost-effective approach for sensitive detection of DNA.2. Label-free and ultrasensitive chemiluminescence detection of DNA based on Exonuclease Ⅲ-assisted cascaded recycling amplification strategyIn this work, a very simple, label-free, isothermal, and ultrasensitive chemiluminescence DNA biosensor has been developed on the basis of an Exo Ⅲ-assisted cascaded recycling amplification strategy. A duplex DNA probe constructed by the hybridization of a quadruplex-forming oligomer with a molecular beacon (MB) is ingeniously designed. Upon sensing of the analyte nucleic acid, the strand of MB in the duplex DNA probe could be stepwise removed by Exo III accompanied by the releasing of target DNA and autonomous generation of secondary target DNA fragment for the successive hybridization and cleavage process. Simultaneously, numerous quadruplex forming oligomers are liberated and folded into G-quadruplex-hemin complexes with the help of K+and hemin to stimulate the generation of CL in the presence of H2O2and luminol. Because of this cascaded recycling amplification and the specifically catalyzed formation of G-quadruplex-hemin complexes, this newly designed protocol provides an ultrasensitive CL detection of DNA down to the8fM level, can discriminate mismatched DNA from perfectly matched target DNA, and holds a great potential for early diagnosis in gene-related diseases. It further could be developed as a universal protocol for the detection of various DNA sequences and may be extended for the detection of aptamer-binding molecules.