Exonuclease III-Assisted Cascade Signal Amplification Strategy for Label-Free and Ultrasensitive Chemiluminescence Detection of DNA

Detection of ultralow concentrations of specific nucleic acid sequences is a central challenge in the early diagnosis of genetic diseases and biodefense applications. Herein, we report a simple and homogeneous chemiluminescence (CL) method for ultrasensitive DNA detection. It is based on the exonuclease III (Exo III)-assisted cascade signal amplification and the catalytic effect of G-quadruplex-hemin DNAzyme on the luminol-H2O2 CL system. A quadruplex-forming DNA probe hybridizes a hairpin DNA probe to construct a duplex DNA probe as recognition element. Upon sensing of target DNA, the recognition of target DNA and the duplex DNA probe triggers the Exo III cleavage process, accompanied by releasing target DNA and generating a new secondary target DNA fragment. The released target DNA and the secondary target DNA are recycled. Simultaneously, numerous quadruplex-forming sequences are liberated and bind hemin to yield G-quadruplex hemin DNAzyme, which subsequently catalyze the luminol-H2O2 reaction to produce strong CL emission. This method exhibited a high sensitivity toward target DNA with a detection limit of 8 fM, which was about 100 times lower than that of the reported DNAzyme-based colorimetric system for DNA detection with Exo III-assisted cascade signal amplification. This method provides a simple, isothermal, and low-cost approach for sensitive detection of DNA and holds a great potential for early diagnosis in gene-related diseases.