Tri-Mode CRISPR-Based Biosensor for miRNA Detection: Enhancing Clinical Diagnostics with Cross-Validation

作者：Jiangbo Dong, Wenhao Ma, Shiying Zhou, Xinyao Li, Liyuan Deng, Changjun Hou, Danqun Huo · 发表于：Analytical Chemistry · 年份：2025 · DOI：10.1021/acs.analchem.5c02348 · 被引用次数：18 · 研究领域：Advanced biosensing and bioanalysis techniques、CRISPR and Genetic Engineering、Biosensors and Analytical Detection

In vitro diagnostics require the accurate detection of disease-associated target biomolecules at ultralow concentrations. A multimode sensing strategy is considered as a potential method for in vitro diagnosis because it allows cross-validation of test results through data complementation and self-calibration, and provides double confirmation. Here, we present a CRISPR/Cas12a-powered trimode biosensor (CPTMB) for ultrasensitive and reliable analysis of miRNA. Briefly, the presence of target miRNA initiates rolling circle extension-driven loop-mediated isothermal amplification (R-LAMP), which subsequently activates the trans-cleavage activity of CRISPR/Cas12a. Then, the hairpin probe (HP) biogate on nucleic acid-functionalized MB@Fe-MOF signal probe was degraded by Cas12a, leading to the release of methylene blue (MB) signal molecules encapsulated within Fe-MOF nanocarriers. Due to the capability of MB to generate output responses across three distinct modes: electrochemical (EC), fluorescence (FL), and ultraviolet-visible spectroscopy (UV-vis), a trimodal sensing system is achieved. Benefiting from the efficient signal amplification capabilities of R-LAMP and CRISPR/Cas12a, this strategy enables rapid detection of target miRNA at femtomolar levels within 70 min. Furthermore, the detection results across the three modes cross-validate one another, thereby enhancing the reliability of the analysis. More importantly, the platform has been successfully applied to miRNA analysis i...