Nanomaterial-enabled CRISPR-Cas biosensing for non-nucleic acid targets: Strategies, mechanisms, and applications

Nanomaterial-assisted CRISPR-Cas biosensing has transcended nucleic acid detection, emerging as a prominent paradigm for the ultrasensitive analysis of non-nucleic acid targets. This review systematically elucidates recent advances by proposing a novel three-dimensional framework encompassing transduction efficiency, signal output, and application adaptation. We first focus on molecular engineering approaches that convert non-nucleic acid recognition events into CRISPR activation signals, critically assessing the inherent efficiency limits and bottlenecks of key techniques such as aptamer switches, nuclease allosteric effects, substituent steric hindrance, Cas protein allosteric regulation, and Cas protein engineering. We then detail how various nanomaterials, including AuNPs, QDs, UCNPs, MOFs, nanozymes, synergistically enhance the efficiency of optical, electrochemical, and multimodal signal output. Utilizing this framework, the review analyzes representative applications in detecting proteins, small molecules, and heavy metal ions, clarifying the mechanisms behind performance enhancement and current limitations. Finally, we discuss persistent challenges and prospectively proposes disruptive future directions, such as constructing CRISPR sensing networks, developing intelligent nanocarriers, and creating closed-loop theranostic systems. This study provides a technical overview, a critical framework, and a design roadmap, facilitating the rational design of biosensors and enabling the realization of integrated bio-regulatory platforms.