Versatile biosensing platform for DNA detection based on a DNAzyme and restriction-endonuclease-assisted recycling

On the basis of a DNAzyme and a restriction-endonuclease-assisted target recycling strategy using Pd-Au alloy nanocrystals to immobilize probe DNA on an electrode and catalyze the reduction of H₂O₂ which amplified signal and promoted the detection sensitivity, a versatile biosensing platform for DNA detection was proposed. Using p53 and oral cancer genes as models, hemin/G-quadruplex simultaneously acted as a reduced nicotinamide adenine dinucleotide (NADH) oxidase and a horseradish peroxidase (HRP)-mimicking DNAzyme, and a versatile DNA biosensor was designed for the first time based on the good electrocatalytic activity of Pd-Au alloy nanocrystals. Hemin/G-quadruplex catalyzed the reduction of H₂O₂, which was generated from NADH in the presence of O₂, to produce an electrochemical signal when thionine functioned as the electron mediator. Moreover, the nicking endonuclease N.BstNB I caused the target DNA to cycle for multiple rounds and further amplified the electrochemical response. This versatile DNA biosensor exhibited linear ranges for the detection of p53 and oral cancer genes from 0.1 fmol L^-1 to 0.1 nmol L^-1 and 0.1 fmol L^-1 to 1 nmol L^-1, respectively. The detection limits, established as 3σ, were estimated to be 0.03 and 0.06 fmol L^-1 for the p53 and oral cancer genes, respectively. The as-prepared biosensor could discriminate mismatched sequences, indicating a satisfactory selectivity and validating the feasibility of the proposed strategy. More importantly, simply by changing the helper DNA, this versatile DNA biosensor could detect different target DNA species, which could create a new avenue for the potential diagnosis of cancer.