Toehold and Hairpin Assembly-Mediated Tripedal DNA Walker for Circulating Tumor DNA Detection in Human Blood

Circulating tumor DNA (ctDNA) levels in the peripheral blood are correlated with tumor burden and malignant progression. Sensitive and accurate detection of ctDNA in human blood remains to be explored. This study introduces a novel approach that employs toehold-assisted ctDNA capture and hairpin assembly-mediated tripedal DNA walker, facilitating the sensitive detection of a broad range of ctDNAs. Specifically, the design includes a capture probe featuring an overhanging toehold domain for the identification of ctDNA, alongside three hairpin structures that incorporate blocked DNAzymes for the DNA walking mechanism. The capture probe identifies ctDNA through a toehold-mediated strand displacement reaction, which subsequently initiates the assembly of the three hairpin structures, resulting in the formation of a tripedal DNA walker. This tripedal walker is capable of initiating a walking process that generates detectable fluorescent signals for the quantification of ctDNA. The toehold-assisted ctDNA capture method demonstrates excellent accuracy and specificity for identical strands with a single-base mismatch, while the tripedal walker exhibits enhanced efficiency compared to bipedal and unipedal walkers. Furthermore, two stages of signal amplification were achieved, allowing for the sensitive detection of ctDNA with a detection limit of 0.3 fM in buffer solutions and 0.8 fM in serum samples. The quantitative analysis of ctDNA in human blood was effectively performed, demonstrating the method’s ability to distinguish between blood samples from healthy individuals and those from patients with tumor. Therefore, this method represents a significant advancement in ctDNA detection for clinical noninvasive liquid biopsies and holds considerable promise for tumor diagnosis.