A versatile homogeneous chemiluminescence biosensing platform based on exonuclease-assisted hybridization chain reaction

Herein, exonuclease III (Exo III)-assisted target recycling amplification is integrated into hybridization chain reaction (HCR) to construct a versatile cascade amplification biosensing platform of Exo III-assisted HCR (Exo-HCR) for label-free chemiluminescence (CL) detection of nucleic acid (target DNA) and protein (lysozyme). This system consists of three molecular beacons (MB1, MB2 and MB3) and one single-stranded target DNA as initiator. In the presence of initiator, MB1 is opened and forms a double-stranded DNA (dsDNA) structure with a blunt 3′ terminus, resulting in the partial digestion of MB1 from its 3′ terminus and the release of target DNA to initiate another cycle. Meanwhile, the released single-stranded region of MB1 initiates HCR through cross-opening of MB2 and MB3, leading to the formation of DNA polymer wires consisting of numerous G-quadruplex units, which are further assembled into hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzymes in the presence of hemin to catalyze the generation of CL signal through the oxidation of luminol by H2O2. This assay allows the amplified label-free CL biosensing of target DNA with a limit of detection (LOD) down to 5.7 fM and excellent specificity to distinguish one-base mismatched target DNA. This method also demonstrates good performance in real sample analysis. Moreover, based on specific recognition of aptamers, the proposed strategy is flexibly applied to sensitive and selective detection of lysozyme, achieving a LOD of 4.2 fM. Therefore, this versatile biosensing platform holds great promise in the fields of biochemical analysis and clinical diagnosis.