CRISPR-Cas12a-Based Nucleic Acid Amplification-Free DNA Biosensor via Au Nanoparticle-Assisted Metal-Enhanced Fluorescence and Colorimetric Analysis.

Cell-free DNA (cfDNA) has attracted significant attention due to its high potential to diagnose diseases, such as cancer. Still, its detection by amplification method has limitations because of false-positive signals and difficulty in designing target-specific primers. CRISPR-Cas-based fluorescent biosensors have been developed but also need the amplification step for the detection. In this study, for the first time CRISPR-Cas12a based nucleic acid amplification-free fluorescent biosensor was developed to detect cfDNA by a metal-enhanced fluorescence (MEF) using DNA-functionalized Au nanoparticle (AuNP). Upon activating the CRISPR-Cas12a complex by the target cfDNA and subsequent single-strand DNA (ssDNA) degradation between AuNP and fluorophore, MEF occurred with color changes from purple to red-purple. Using this system, breast cancer gene-1 (BRCA-1) can be detected with very high sensitivity in 30 min. This rapid and highly selective sensor can be applied to measure other nucleic acid biomarkers such as viral DNA in field-deployable and point-of-care testing (POCT) platform.