Enzyme-Free Crispr/Cas12a Microrna Assay by Amplified Single Particle Analysis

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based detection platforms have increasingly been applied for microRNA (miRNA) detection, especially gained high sensitivity and wide application by combining enzyme-based nucleic acid pre-amplification strategies. Despite great success, the introduction of additional enzymes could result in negative interference between primer residues, higher costs, and non-specific amplification which could sometimes lead to false-positive outcomes or even deactivate the CRISPR system. To address these challenges, an enzyme-free amplified CRISPR/Cas12a miRNA assay was designed by combining the hybridization chain reaction (HCR) amplification and single particle analysis strategy. HCR amplification utilizes the self-assembly properties of nucleic acids, which avoids using any enzyme, enabling efficient amplification in mild conditions. Besides, the single particle analysis by inductively coupled plasma mass spectrometry (Sp-ICPMS) can provide a direct signal amplification, by analyzing individual nanoparticles that retain a great number of metal atoms. Herein, target miRNAs could initiate the HCR amplification circuit, and be converted into double-strand DNAs (dsDNAs) with PAM regions that can be recognized by the CRISPR/Cas system. The activated Cas12a would efficiently sabotage the orthogonal nanoparticles-cross-linking system, which could generate various nano-aggregations with different detectable Sp-ICPMS intensities. Eventually, fM level limit-of-detections (LODs) with excellent selectivity were achieved. Serum-spiked recovery tests were conducted with promising results. To our knowledge, this is the first report of the HCR-CRISPR/Cas12a-based single particle analysis strategy for miRNA detection.