Abstract
Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer-related mortality globally, primarily due to delayed diagnosis at advanced, untreatable stages. Serum microRNA-21 (miR-21) has emerged as a promising non-invasive biomarker for early-stage HCC; however, traditional detection methods such as quantitative real-time PCR (qRT-PCR) require sophisticated equipment and lengthy protocols that hamper point-of-care (POC) deployment. Here, we report the development of a rapid, ultra-sensitive, and highly specific point-of-care diagnostic platform combining reverse transcription-recombinase polymerase amplification (RT-RPA) with CRISPR-Cas12a trans-cleavage activity for serum miR-21 detection. By engineering target-specific crRNA and optimizing a short DNA stem-loop adapter to enable efficient RT-RPA under mild isothermal conditions (37–42 °C), our assay achieves a limit of detection of 3.2 fM within 45 minutes. The trans-cleavage activity of Cas12a is monitored via a fluorophore-quencher single-stranded DNA reporter, delivering strong fluorescent signals discernible visually under ultraviolet light. The assay demonstrated robust specificity, effectively discriminating target miR-21 from single-base mismatched variants and other circulating microRNA family members. Clinical validation using serum samples from early-stage HCC patients, liver cirrhosis controls, and healthy individuals yielded an area under the receiver operating characteristic curve (AUC) of 0.945, significantly outperforming conventional alpha-fetoprotein (AFP) screening (AUC 0.682). This CRISPR-Cas12a-assisted isothermal platform offers a powerful, decentralized diagnostic solution for early liver cancer screening in resource-limited settings.
