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

Abstract

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.