Digital Hydrogel Fluorescent-Enhancing Microspheres via CRISPR/Cas12a for Amplification-Free Relative Quantification of Nucleic Acids.

CRISPR/Cas systems hold great promise for molecular diagnostics, but their amplification-free applications are hampered by weak signals and poor quantification. Here, we developed CrisprDEM, a CRISPR/Cas12a-based digital hydrogel fluorescent-enhancing microsphere system that integrates hydrogel microsphere confinement, microfluidic digital imaging, and machine learning for ultrasensitive and quantitative nucleic acid detection without amplification. Hydrogel microspheres (HMs) efficiently captured and spatially concentrated CRISPR/Cas12a reaction reporters, achieving a 1000-fold signal amplification compared with homogeneous methods. The design of the microfluidic chip arranged the microspheres into a single-layer array, making each microsphere an independent digital reporting unit. The Intelligent Bead Analysis Software enabled automatic analysis and relative quantification via a positive bead ratio (PBR). As a proof-of-concept, we selected the respiratory adenovirus as the detection target. We optimized the CRISPR/Cas12a-microsphere enrichment reaction system and characterized the morphologies and chemical properties of the microspheres before and after enrichment. The results demonstrated that CrisprDEM technology exhibited a detection sensitivity of 10 aM for respiratory adenovirus, exhibiting no cross-reactivity with other respiratory viruses, indicating a high specificity. In the validation of 20 clinical samples, the detection results were consistent with the gold-standard real-time quantitative polymerase chain reaction (qPCR), and the PBR value showed a good linear relationship with the cycle threshold (Ct) value, enabling a relative quantification. This system expands the toolbox for amplification-free CRISPR diagnostics and holds the potential for point-of-care and early infection detection.