Single‐Droplet Dual‐Target Quantification of circRNA Biomarkers for Colorectal Cancer Screening

Circular RNAs (circRNAs) play a crucial role in the oncogenesis, progression, and chemoresistance of tumors. Here, two plasma circRNA biomarkers, circCK1γ3 and circWDR37 is identified and validated. Simultaneous analysis of these biomarkers has the potential to significantly improve the sensitivity of colorectal cancer (CRC) screening. Leveraging the distinct base-cutting preferences of these two Cas proteins, it is successfully expressed and purified LwaCas13a and PsmCas13b, and developed a one-pot Cas13a/13b-RPA assay for the simultaneous detection of circCK1γ3 and circWDR37. To achieve precise detection of circCK1γ3 and circWDR37, the Casµchip, which integrates droplet generation and digital quantification functionalities is designed and fabricated. The Casµchip enables the simultaneous detection of circCK1γ3 and circWDR37 within 70 min. This method exhibits excellent repeatability and high specificity, with a detection limit of 100 copies/reaction. The correlation coefficients for circWDR37 and circCK1γ3 detected by Casµchip and qPCR are 0.9113 and 0.8933, respectively. Casµchip analysis is conducted on a cohort of 60 clinical samples, and the findings indicate that this methodology holds promise for the early detection of colorectal cancer. Casµchip represents the profound integration of CRISPR diagnostics and microfluidic technology, offering a novel paradigm characterized by rapid, precise, and user-friendly features for molecular diagnosis.