Nucleic acid amplification-free biosensor for sensitive and specific cfDNA detection based on CRISPR-Cas12a and single nanoparticle dark-field microscopy (DFM) imaging

Abstract

Circulating cell-free DNA (cfDNA) is known to be an attractive biomarker for liquid biopsy. In this work, a rapid and sensitive biosensor was achieved for cfDNA detection based on CRISPR-associated nuclease 12a (CRISPR-Cas12a) and single nanoparticle dark-field microscopy (DFM) imaging. The biosensor employed gold nanoparticles (AuNPs) as signal source, DFM as readout system, and Meanshift algorithms as the image processing systems, respectively. The presence or absence of cfDNA caused the different existing states (monomer or aggregated state) of AuNPs. AuNPs monomers could be effectively distinguished from the aggregated ones under DFM since the AuNPs aggregation could induced the green-to-yellow or green-to-red changing of scattering color. The monomer ratio easily obtained by Meanshift and partial least-square (PLS) algorithms was used for quantitative analysis. Using this strategy, breast cancer gene-1 (BRCA-1), a representative of cfDNA biomarker for breast cancer could be measured with high sensitivity in 40 min and has a low detection limit of 0.081 fM. It is hoped that this nucleic acid amplification-free sensor could be also utilized to detect other nucleic acid biomarkers, and thus, provides a novel strategy for biomedical image analysis.