Amplification-Free Electrochemiluminescent Biosensor for Ultrasensitive Detection of <i>Fusobacterium nucleatum</i> Using Tetrahedral DNA-Based CRISPR/Cas12a

<i>Fusobacterium nucleatum</i>, a bacterium linked to colorectal cancer, possesses a specific gene called <i>fadA</i> that serves as an early diagnostic biomarker. The CRISPR/Cas12a system has demonstrated marked potential for nucleic acid detection due to its satisfactory selectivity and trans-cleavage ability. However, most CRISPR/Cas-based sensors suffer from problems such as probe entanglement or local aggregation, reducing the Cas enzyme efficiency. In this study, an amplification-free biosensing platform for ultrasensitive detection of <i>F. nucleatum</i> was developed by integrating the highly specific CRISPR/AsCas12a with an improved electrochemiluminescence (ECL) biosensor. Different from the conventional 1- or 2-dimensional probes, the platform was constructed by tetrahedral DNA nanostructure (TDN) probes conjugated with quenchers and coralliform gold (CFAu) functionalized with luminescent agents. The TDN serves as an exceptional scaffold to modulate the recognition unit, substantially enhancing the recognition and cleavage efficiency of AsCas12a toward the probes. Furthermore, the high surface area of CFAu provides extensive landing sites for the luminescent agents, thereby improving the detection sensitivity. The prepared ECL biosensor exhibited a wider linear range (10 fM to 100 nM) and was capable of detecting <i>F. nucleatum</i> down to 1 colony-forming unit/ml. Additionally, the high mismatch sensitivity of AsCas12a to protospacer adjacent motifs and nearby areas provides a strategy for distinguishing mutant from wild-type sequences. Finally, by designing CRISPR RNA (crRNA), this diagnostic method can also be easily modified to detect other bacteria or biomarkers for the early diagnosis of various diseases.