An enzyme-free amplification strategy for sensitive assay of circulating tumor DNA based on wheel-like catalytic hairpin assembly and frame hybridization chain reaction

•An enzyme-free amplification strategy for sensitive assay of circulating tumor DNA based on WCHA and FHCR is proposed.•FHCR can fold up more compact and dense DNA nanostructures to shorten the distance between electrochemical species and the electrode interface.•This strategy provides a versatile platform for ctDNA detection and advances the field of DNA self-assembly.•This proposed method is applied for clinical sample analysis with high sensitivity and specificity for the diagnosis of breast tumors. As an ideal liquid biopsy biomarker candidate, circulating tumor DNA (ctDNA) plays an important role in the non-invasive diagnosis of cancer. Sensitive and precise analysis of ctDNA plays a vital role in promoting clinical applications. Herein, we have developed a novel electrochemical biosensor for ctDNA assay coupling wheel-like catalytic hairpin assembly (WCHA) and frame hybridization chain reaction (FHCR). The ctDNA bound to the DNA transducer by the toehold strand displacement and released the initiator to promote the recycle amplification of WCHA, which lead to the formation of a great number of wheel-like DNA products hybridized with the capture probes immobilized on the gold electrode surface. In addition, the metastable dumbbell probes (DSH1 and DSH2) are designed for hybridization chain reaction. Compared with the traditional HCR, this novel form of HCR (FHCR) can fold up more compact and dense DNA nanostructures, which greatly reduce the space potential resistance and enhance the electronic transmission ability. The combination of dual signal amplification strategies (WCHA/FHCR) significantly improve the sensitivity of ctDNA detection, creating a limit of detection (LOD) as low as 8.3 fM. More importantly, the proposed method is successfully applied in the analysis of clinical samples, and has an effective classifier with an AUC of 0.909, showing a sensitivity of 86.7 % and a specificity of 80.0 % for the diagnosis of breast tumors. Therefore, this platform has great potential in bioanalysis and clinical diagnostics.