Entropy-driven self-assembled enzyme-DNA nanomatrix cascade DNAzyme-CRISPR/cas system for multiplexed enhancement of...

Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated Proteins (CRISPR/Cas) system can accurately identify and cleave target DNA sequences, while the effective combination of DNA nanomatrix and entropy-driven self-assembled enzymes can significantly enhance the sensitivity, stability, and diversified functionality of sensors through highly ordered molecular arrangement and spontaneous efficient assembly processes. Herein, a carbon-encapsulated MoS₂ hollow nanorod (C-MoS₂) with excellent conductivity and multiple active sites is used to construct bioanode of biofuel cell by integrating it with an entropy-driven self-assembled enzyme-DNA nanomatrix cascade DNAzyme-CRISPR/Cas system. When thrombin binds aptamer, it exposes the trigger strand on the anode, initiating chain displacement. This activates DNAzyme, triggering a cascade reaction that cleaves and releases the probe strand. The probe then binds CrRNA, forming a multimeric complex with Cas protein.When non-specific cleavage of the single strand occurs, it leads to the release of glucose oxidase (GOD) from the DNA matrix and causes a significant decrease in the value of the system's open-circuit voltage (E^OCV). The E^OCV values show a good negative correlation with the concentration of thrombin (TB) in the range of 0.00001-100 nM, achieving a limit of detection of 3.55 fM (S/N = 3).