Novel electrochemical biosensor based on Exo III-assisted digestion of dsDNA polymer from hybridization chain reaction in homogeneous solution for CYFRA 21-1 DNA assay

A novel electrochemical biosensing strategy was proposed to detect cytokeratin fragment antigen 21-1 (CYFRA 21-1) DNA based on Exo III-assisted digestion of dsDNA polymer (EADDP) from hybridization chain reaction (HCR). Primarily, the presence of target can drive a catalytic hairpin assembly (CHA) reaction, which was aimed to achieve target recognition and circulation. Then the HCR can be triggered for further signal amplification and generate long dsDNA polymer with signal tags. Subsequently, the introduction of Exo III can digest the long dsDNA polymer to produce large amounts of double signal fragments (DSFs). The above experiments were all carried out in homogeneous solution. Finally, the released DSF can be captured onto the electrode directly by capture probe (CP) and a highly amplified electrochemical signal can be detected. The EADDP in homogeneous solution circumvented complex solid-liquid interface reaction and tedious operation steps on electrode. Besides, one target can be converted into abundant DSFs, which greatly improved the sensitivity. This biosensor exhibited a low detection limit (0.0348 fM) and wide linear range (5 fM ∼ 50 nM) for CYFRA 21-1 DNA biosensing with reliable specificity and stability. • A novel strategy based on Exo III-assisted digestion of dsDNA polymer (EADDP) was proposed. • The Exo III was firstly used to digest dsDNA polymer to produce abundant double signal fragments (DSF). • The EADDP can convert one target into large amount of DSF in homogeneous solution. • EADDP in homogeneous solution and direct capture of DSF simplified the operation steps on electrode. • This biosensor achieved a low detection limit of 0.0348 fM for CYFRA 21-1 DNA detection.