A rapid and highly sensitive ctDNA detection platform based on locked nucleic acid-assisted catalytic hairpin assembly circuits

Abstract

As a promising biomarker of liquid biopsy, circulating tumor DNA (ctDNA) plays a paramount role in the early diagnosis of noninvasive cancer. The isothermal catalytic hairpin assembly (CHA) strategy has great potential for in vitro detection of ctDNA in low abundance. However, a traditional CHA strategy for ctDNA detection at the earlier stages of cancer remains extremely challenging, as annoying signal leakage from the ‘breathing’ phenomenon and nuclease degradation occur. Herein, we report a locked nucleic acid (LNA)-incorporated CHA circuit for the rapid and sensitive detection of target ctDNA. The target ctDNA intelligently catalyzed LNA-modified hairpins H₁ and H₂via a range of toehold-mediated strand displacement processes, leading to the continuous generation of an H₁–H₂ hybrid for the amplified fluorescence signal. In comparison to conventional CHA circuits, the stronger binding affinity of LNA–DNA bases greatly inhibited the breathing effect, which endowed it with greater thermodynamic stability and resistance to nuclease degradation in the LNA-assisted CHA system, thus achieving a high signal gain. The developed CHA circuit demonstrated excellent performance during target ctDNA detection, with a linear range from 10 pM to 5 nM, and its target detection limit was reached at 3.3 pM. Moreover, this LNA-assisted CHA system was successfully applied to the analysis of target ctDNA in clinical serum samples of breast cancer patients. This updated CHA system provides a general and robust platform for the sensitive detection of biomarkers of interest, thus facilitating the accurate identification and diagnosis of cancers.