Dual‐miRNA Synchronous Detection Technology Combining Isothermal Amplification and CRISPR/Cas12a System With AND/NOT Logic Gates for Colorimetric‐Fluorescent Signal Output

ABSTRACT Early and accurate diagnosis of cancer relies on the detection of highly sensitive and specific biomarkers. MicroRNAs (miRNAs), as key regulatory factors, are ideal diagnostic markers for cancer. However, their extremely low abundance and susceptibility to degradation in biological samples pose significant challenges for traditional detection methods. To address this challenge, this study developed a dual‐mode synchronous detection biosensor for the combined detection of breast cancer‐related biomarkers miRNA‐96 and miRNA‐21. The biosensor integrates an AND‐logic‐gate colorimetric detection (CRISPR/Cas12a‐nanozyme) with a NOT‐logic‐gate fluorescence detection (RCA‐based), enabling multiplexed target discrimination. The entire system forms a dual‐input (miRNA‐96, miRNA‐21), triple‐output (colorimetric, FAM, ROX) molecular logic circuit. Its output patterns are: (1,1) to (1,0,0); (1,0) to (0,0,1); (0,1) to (0,1,0); (0,0) to (0,1,1). The biosensor demonstrated high performance, with colorimetric detection limits of 29.70 fM (miRNA‐96) and 47.81 fM (miRNA‐21), and fluorescence limits of 103.54 fM and 366.06 fM, respectively. It exhibits outstanding specificity against similar miRNA sequences, demonstrates good stability, and achieves accurate recovery rates in clinical serum samples, enabling reliable differentiation between plasma samples from healthy individuals and breast cancer patients. This cost‐effective, modular strategy provides a sensitive tool for early breast cancer screening.