A novel enzyme-free electrochemical sensing strategy was proposed for sensitive monitoring of DNA and miRNA by smart combination of the cyclic cleavage reaction of Mg2+-dependent DNAzyme and the host-guest inclusion between ferrocene-labeled hairpin probe (H-1) and nitrogen-doped reduced graphene oxide/β-cyclodextrin polymer (NRGO/β-CDP) nanocomposites. The synthesized NRGO/β-CDP nanocomposites with high electrocatalytic activity and recognition capability were modified on the glassy carbon electrode to construct the sensing platform. Upon the hybridization reaction of subunit DNA in the loop region with target sequence, the active DNAzyme was liberated from the caged structure, which bound with H-1 to catalyze its cleavage in the presence of Mg2+ and triggered the target recycling amplification for the cleavage of a large number of H-1. Each cleaved H-1 was divided into two single-stranded oligonucleotides, leading to an obvious enhancement of peak current by the molecular recognition of β-CDP on the electrode. Thus, the constructed biosensor showed high sensitivity and selectivity for DNA and miRNA assays, with wide concentration ranges of 0.01-1000 and 0.05-500 pM and low detection limits of 3.2 and 18 fM, respectively. This developed sensing strategy may become a promising nucleic acid detection method in bioassays and clinical diagnosis.
Keywords: DNAzyme; biosensor; electrochemical detection; nitrogen-doped reduced graphene oxide; β-cyclodextrin polymer.