A triple-cycle amplification biosensor for colorimetric detection of mutant PIK3CAE545K based on cascade-driven DNA walker and branched hybridization strand reaction

Circulating tumor DNA (ctDNA) is an ideal candidate for liquid biopsy biomarkers. Therefore, detecting a low abundance of ctDNA is essential for early cancer diagnosis. Here, we developed a novel triple circulation amplification system integrating entropy and enzyme cascade-driven three-dimensional (3D) DNA walker and branched hybridization strand reaction (B-HCR) for ultrasensitive detection of breast cancer-related ctDNA. In this study, the 3D DNA walker was constructed by inner track probes (NH) and complex S on a microsphere. Once the DNA walker was triggered by the target, the strand replacement reaction ran first and kept circulating to rapidly displace the DNA walker containing 8-17 DNAzyme. Secondly, the DNA walker could repeatedly cleave NH autonomously along the inner track, generating numerous initiators, and then promoting B-HCR to activate the third cycle. Subsequently, the split G-rich fragments were brought in close to form the G-quadruplex/hemin DNAzyme by adding hemin, with the addition of H2O2 and ABTS, the target could be observed. Benefiting from triplex cycles, the PIK3CAE545K mutation detection possesses a good linear range from 1-103 fM, and the limit of detection was 0.65 fM. Due to the low cost and high sensitivity, the proposed strategy has great potential in early diagnosis of breast cancer.