CRISPR-Cas12a and DNA Tetrahedron Assemblies Amplified Fluorescence Anisotropy for the Sensitive Detection of Hepatitis B Virus DNA

Fluorescence anisotropy (FA) has been widely used for analyzing biomolecules due to its high throughput, homogeneous detection, and strong resistance to photobleaching. However, the traditional FA method suffers from low sensitivity when the target molecules are small and rotate rapidly, often producing insignificant changes in the FA value. In this study, by combining double signal amplification through the trans-cleavage of CRISPR-Cas12a and DNA tetrahedron assemblies with a large molecular size, a new, fast, simple and highly sensitive FA method was constructed to achieve the quantitative detection of hepatitis B virus DNA (HBV-DNA). The experimental results showed that the linear range of this method was 0.5–9 nmol/L, and the detection limit (LOD = 3σ/k) was 48 pmol/L. In addition, the method demonstrated excellent selectivity and anti-interference, and it was successfully applied to detect HBV-DNA in human serum, indicating that this method has the potential for clinical diagnosis.