Fusobacterium nucleatum, a bacterium linked to colorectal cancer, possesses a specific gene called fadA 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. "Electrochemiluminescence (ECL) has emerged as a sensitive method for detecting small molecules, nucleic acids, and proteins, due to its superior spatiotemporal control and virtually undetectable background noise. In ECL, luminescent reactants on the electrodes are regenerated through electrochemical reactions, allowing them to repeatedly participate in ECL reactions with remaining coreactants. This ongoing photon production during each measurement cycle substantially enhances the sensitivity of ECL." said study author Jieling Qin, a professor at Beijing Institute of Technology. Hence, in this study, the authors developed an amplification-free biosensing platform for ultrasensitive detection of F. nucleatum by integrating the highly specific CRISPR/AsCas12a with an improved 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 efciency 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 F. nucleatum 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." said Xindan Zhang.
The electrochemical sensing platform based on the CRISPR/Cas system serves as a reliable detection method for F. nucleatum and can be tailored for detecting other nucleic acids and pathogens by rationally designing crRNA sequences. 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.
Authors of the paper include Xindan Zhang, Minkang Wu, Haoran Shi, Soochan Kim, Shixiang Lu, Ping Wang, and Jieling Qin.
This work was supported by the China Postdoctoral Science Foundation (2023M730260), Postdoctoral Fellowship Program of CPSF (GZC20233413), and Beijing Institute of Technology Research Fund Program for Young Scholars programs.
The paper, "Amplifcation-Free Electrochemiluminescent Biosensor for Ultrasensitive Detection of Fusobacterium nucleatum Using Tetrahedral DNA-Based CRISPR/Cas12a" was published in the journal Cyborg and Bionic Systems on May 1, 2025, at DOI: 10.34133/cbsystems.0266.
