CRISPR/Cas12a Assisted Branched Hybridization Chain Reaction (bHCR) for the Sensitive Fluorescent Determination of Mucin 1 (MUC1)

Sensitive and early-stage detection of tumor biomarkers is a key factor in improving cancer diagnostic accuracy and prognostic evaluation. Mucin 1 (MUC1), an overexpressed transmembrane glycoprotein in various epithelial cancers, is a significant clinical biomarker. This paper presents a fluorescent method based on an aptamer-triggered branched hybridization chain reaction (bHCR) and CRISPR/Cas12a signal output was developed for the determination of mucin 1 (MUC1). The approach leverages the specific binding of MUC1 to an aptamer-containing hairpin probe, initiating a cascade bHCR process that forms highly branched double-stranded DNA nanostructures enriched with protospacer adjacent motif (PAM) sites and target sequences. These DNA products activate the Cas12a/crRNA complex’s trans-cleavage activity, effectively cleaving a fluorophore-quencher labeled ssDNA reporter and enhancing fluorescence output. Compared with linear HCR alone, the branched architecture provides a larger number of Cas12a-activating duplexes per recognition event and improves signal gain. Under the optimal conditions, the sensor offers a broad linear detection range from 1 fg mL−1 to 1 ng mL−1 and a low detection limit of 0.54 fg mL−1. Additionally, the method showed good selectivity against carcinoembryonic antigen, alpha-fetoprotein, immunoglobulin G, and pepsinogen I. Preliminary recovery experiments performed in 50-fold diluted human serum further supported the analytical feasibility of the method. This work presents a proof-of-concept integration of aptamer-triggered bHCR with Cas12a-mediated fluorescent transduction for sensitive protein analysis.