An ultra-sensitive electrochemical biosensor for circulating tumor DNA utilizing dual enzyme-assisted target recycle and hybridization chain reaction amplification strategies

Circulating tumor DNA (ctDNA) has emerged as a pivotal biomarker for cancers, offering a non-invasive means of detection through liquid biopsies. However, the current methodologies for ctDNA detection often struggle with sensitivity, especially when confronted with ultra-low concentrations. To address this challenge, we have engineered a remarkably sensitive electrochemical biosensor that can detect low concentration of ctDNA. This breakthrough is achieved by integrating a dual enzyme-assisted amplification mechanism with hybridization chain reaction (HCR). In the initial phase, the presence of ctDNA triggers the unfolding of the H2 hairpin structure, resulting in a segment of double-stranded DNA. The subsequent introduction of Klenow (3 '-> 5 ' exo-) and nicking endonuclease (Nb.BbvCI) enzymes leads to the abundant production of extended DNA (EXTDNA). This step critically enhances the initial amplification of the target DNA, ensuring superior specificity and selectivity thanks to the synergistic enzymatic activity. EXTDNA efficiently unfolds the H1 hairpin structure immobilized on the gold electrode, which acts as the initiation point for the HCR. Further amplification of the trace target DNA is achieved through an additional H3 and H4 hybrid-mediated HCR. Finally, the differential pulse voltammetry signal of methylene blue is increased significantly. Our results reveal that the developed electrochemical biosensor displays an exceptional linear correlation with ctDNA across concentrations ranging from 10 fM to 20 pM, with an unprecedented detection limit of 2.3 fM (3 s/k). This innovative approach shows immense potential for the ultrasensitive detection of ctDNA, promising broad applications in early cancer detection and monitoring.