A versatile label-free electrochemical biosensor for circulating tumor DNA based on dual enzyme assisted multiple amplification strategy

A versatile label-free electrochemical biosensor based on dual enzyme assisted multiple amplification strategy was developed for ultrasensitive detection of circulating tumor DNA (ctDNA). The biosensor consists of a triple helix molecular switch (THMS) as molecular recognition and signal transduction probe, ribonuclease HII (RNase HII) and terminal deoxynucleotidyl transferase (TdT) as dual enzyme assisted multiple amplification accelerator. The presence of target ctDNA could open THMS and trigger RNase HII-assisted homogenous target recycling amplification to produce substantial signal transduction probe (STP). The released STP hybridized with the capture probe immobilized on a gold electrode, then TdT and assistant probe were further employed to fulfill TdT-mediated cascade extension and generate stable DNA dendritic nanostructures. The electroactive methyl blue (MB) was finally used as the signal reporter to realize the multiple electrochemical amplification ctDNA detection as the amount of MB is positively correlated with the target ctDNA. Combined with the efficient recognition capacity of the designed THMS and the excellent multiple amplification ability of RNase HII and TdT, the constructed sensing platform could detect KRAS G12DM with a wide detection range from 0.01 fM to 1 pM, and the limit of detection as low as 2.4 aM. Besides, the platform is capable of detecting ctDNA in biological fluid such as plasma. More importantly, by substituting the loop of THMS with different sequences, this strategy could be conveniently expanded into the detection of other ctDNA, showing promising potential applications in clinical cancer screening and prognosis.