Ultrasensitive miRNA-21 detection via catalytic hairpin assembly-enhanced light-initiated chemiluminescence for early cancer diagnosis.

MicroRNAs (miRNAs) are promising molecular markers for early-stage cancer, enabling advancements in early diagnosis, precision treatment, and prognosis evaluation. However, their detection remains challenging due to low abundance, driving the demand for highly sensitive and accurate sensing platforms. Herein, we developed a cascade amplification strategy integrating catalytic hairpin assembly (CHA) with a light-initiated chemiluminescent assay (LiCA) for ultrasensitive detection of serum miRNA-21. The platform employs H1-functionalized chemibeads (¹O₂-luminescent acceptors) and H2-conjugated sensibeads (photosensitizer donors), wherein target miRNA triggers ternary complex formation and initiates autonomous CHA cycling. This process facilitates femtomolar-sensitive, low-background detection without RNA extraction by utilizing proximity-driven (<200 nm) singlet oxygen transfer to generate a collective chemiluminescence signal. The assay exhibits high sensitivity (LOD: 0.21 pM) and excellent linearity (R² = 0.9958), coupled with strong matrix tolerance in 2 % human serum, as evidenced by accurate and consistent recoveries (98.8-112 %) with <8 % deviation at spiked concentrations from 0.1 to 5 pM. When applied to clinical serum samples (n = 78), the platform demonstrated perfect diagnostic discrimination (AUC = 1.00) with 100 % sensitivity and specificity at a cutoff of 0.667 pM across breast, gastric, and colorectal cancers. A strong correlation with qPCR (r = 0.78) was observed, and miRNA-21 levels were significantly elevated in cancer patients compared to healthy controls. This platform combines the programmability of CHA with the anti-interference advantages of LiCA, offering a robust tool for low-abundance miRNA analysis in complex matrices.