Tetrahedral DNA nanostructures-assisted electrochemical assay for detecting circulating tumor DNA by combining a masking tactic with 3D-hybridization chain reactions.

Circulating tumor DNA (ctDNA) is a remarkable noninvasive tumor marker that plays a crucial role in tumor diagnosis, prognosis and treatment. However, detecting low-abundance ctDNA from a substantial amount of nucleic acids originating from healthy cells is challenging. Herein, we proposed a tetrahedral DNA nanostructures (TDNs)-assisted electrochemical biosensor for ctDNA detection. This biosensor combines a masking tactic with 3D-hybridization chain reactions. Masking hairpins (MHs) were initially introduced to prevent interference from wild-type (WT) DNA. Then, the initiator sequence was transferred to the electrode surface modified with TDNs by the target ctDNA. The initiator sequence triggers the 3D self-assembly of hairpin strands, leading to the formation of DNA networks or even DNA hydrogels (long reaction time). This process generates numerous evenly distributed biotin molecules that can bind to streptavidin peroxidase to considerably amplify the signal. This method exhibits high sensitivity (the minimum concentration for detecting ctDNA is 1 aM, which corresponds to 60 ctDNA molecules in 100 μl sample) and excellent specificity (single mismatch). More importantly, this high-performance sensor can detect ctDNA with other mutation sites and their mixtures by modifying the corresponding capture probes on the TDNs. Furthermore, this ultrasensitive sensor effectively detects target ctDNA (0.001 %) at high levels of WT DNA and in complex matrices such as serum. These findings suggest that the sensor has promising potential as a noninvasive tool for early tumor diagnosis.