A sensitive electrochemiluminescence DNA biosensor based on the signal amplification of ExoIII enzyme-assisted hybridization chain reaction combined with nanoparticle-loaded multiple probes

An electrochemiluminescence (ECL) DNA biosensor based on ExoIII exonuclease assistance and hybridization chain reaction (HCR) amplification technology has been constructed. ExoIII exonuclease and triple-helix DNA molecular switch are used in detecting a target in circulation. By combining HCR with AuNPs@DNA, a novel signal probe is built, which enables multiple signal amplification and the high-sensitive detection of transgenic rice BT63 DNA. The Fe₃O₄@Au solution is added to a magneto-controlled glassy carbon electrode, and sulfhydryl-modified capture DNA (CP) is immobilized on Fe₃O₄@Au through the Au–S bond. Mercaptoethanol is added to close sites and prevent the nonspecific adsorption of CP on the magnetron glassy carbon electrode. A target DNA is added to a constructed triple-helix DNA molecular centrifuge tube for reaction. Owing to base complementation and the reversible switching of the triple-helix DNA molecular state, the target DNA turns on the triple-helix DNA molecular switch and hybridizes with a long-strand recognition probe (RP) to form a double-stranded DNA (dsDNA). Exonuclease ExoIII is added to specifically recognize and cut the dsDNA and to release the target DNA. The target DNA strand then circulates back completely to open the multiple triple-helix DNA molecular switch, releasing a large number of signal transduction probes (STP). To hybridize with CP, a large amount of STP is added to the electrode. Finally, a AuNPs@DNA signal probe is added to hybridize with STP. H1 and H2 probes are added for the hybridization chain reaction and the indefinite extension of the primer strand on the probe. Then, tris-(bipyridyl)ruthenium(II) is added for ECL signal detection with PBS-tri-n-propylamine as the base solution. In the concentration range 1.0 × 10⁻¹⁶ to 1.0 × 10⁻⁸ mol/L of the target DNA, good linear relationship was achieved with the corresponding ECL signal. The detection limit is 3.6 × 10⁻¹⁷ mol/L. The spiked recovery of the rice samples range from 97.2 to 101.5%. The sensor is highly sensitive and has good selectivity, stability, and reproducibility. Graphical abstract: