Thermosensitive hydrogel-enhanced RPA-CRISPR/Cas12a biosensor for ultrasensitive detection of methylated loci in breast cancer ctDNA

  • Anal Chim Acta. 2026 Feb 22:1388:345101. doi: 10.1016/j.aca.2026.345101.
Jianxun Hou  1 ,  Yingjie Wang  1 ,  Weiguang Yuan  2 ,  Yajie Gong  1 ,  Yuanyuan Yu  1 ,  Xuquan Qin  1 ,  Hui Li  1 ,  Youxue Zhang  1 ,  Huawen Shi  1 ,  Yanbo Chen  3 ,  Xianyu Zhang  4
Affiliations
  • 1. Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, 150086, China.
  • 2. Institute of Cancer Prevention and Treatment, Harbin Medical University, Harbin, Heilongjiang, 150086, China; Institute of Cancer Prevention and Treatment Heilongjiang Academy of Medical Sciences, Harbin, Heilongjiang, 150086, China.
  • 3. Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, 150086, China. Electronic address: [email protected].
  • 4. Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, 150086, China. Electronic address: [email protected].
PMID: 41611402 DOI: 10.1016/j.aca.2026.345101
Abstract

Background: Methylation differences exist between Breast Cancer tissues and normal tissues. The release of methylated circulating tumor DNA (ctDNA) by tumor cells provides a foundation for Breast Cancer liquid biopsy using methylated ctDNA. However, detection of low-abundance methylated loci in ctDNA remains a significant challenge to date. Existing one-tube detection systems cannot avoid target depletion caused by CRISPR/Cas12a cleavage, leading to reduced sensitivity.

Results: This study developed a thermosensitive hydrogel-based one-tube RPA-CRISPR/Cas12a detection system, combined with methylation-sensitive restriction endonucleases (MSRE), for the detection of specific methylated loci in Breast Cancer ctDNA. This method achieves spatial separation while maintaining connectivity of reaction phases in a single tube for the first time, and the thermosensitive hydrogel does not exert inhibitory effects on either system. The system can specifically recognize methylated target molecules with a limit of detection (LOD) as low as 1 × 10-8 ng/μL (≈70 copies/μL), outperforming the current glycerol-enhanced one-tube reaction system. It is capable of distinguishing methylated fractions as low as 0.05 %, with a sensitivity twice that of the gold standard methylation-specific quantitative PCR (Methylight). Detection of genomic DNA (gDNA) from tumor tissues and paired plasma ctDNA of 15 clinical patients using this method showed both sensitivity and specificity reaching 100 %.

Significance: This novel, highly sensitive, efficient, and portable detection method innovatively resolves the target depletion issue caused by CRISPR/Cas12a cleavage in traditional RPA-CRISPR/Cas12a systems via thermosensitive hydrogel-mediated single-tube phase separation technology. It provides a new technical pathway for the accurate detection of low-abundance methylated circulating tumor DNA (ctDNA), will significantly enhance the level of Breast Cancer liquid biopsy, and offer strong support for the diagnosis and differential diagnosis of Breast Cancer.

Keywords
Biosensor; Breast cancer; Circulating tumor DNA(ctDNA); DNA methylation; Thermosensitive hydrogel.
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