Ultrasensitive miRNA Detection Based on Magnetic Upconversion Nanoparticle Enhancement and CRISPR/Cas13a-Driven Signal Amplification.

ABSTRACT

MicroRNAs (miRNAs), a class of small molecules with important regulatory functions, have been widely used in the field of biosensing as biomarkers for the early diagnosis of various diseases. Therefore, it is crucial to develop an miRNA detection platform with high sensitivity and specificity. Here, we have designed a CRISPR/Cas13-based enzymatic cyclic amplification system and regarded the magnetic upconversion nanoparticles (MUCNPs) as a biosensor of outputting the detection signal for the highly sensitive and high-fidelity detection of miRNAs. MUCNPs were composed of UCNPs (fluorescence donors) and Fe3O4@AuNPs (fluorescence acceptors) through double-stranded DNA hybrid coupling. The target miRNA acted as an activator, which could activate the trans-cleavage activity of Cas13a to the well-designed Trigger containing two uracil ribonucleotides (rU) in its loop and trigger a strand displacement reaction to generate a large amount of single-stranded DNA, resulting in the release of the UCNPs from MUCNPs. Benefiting from the high fidelity and high selectivity of CRISPR/Cas13a, the great effect of triggered enzymatic cycle amplification, and the high-intensity luminescent signal of MUCNPs, this method possessed miRNA detection capability with high sensitivity and specificity even in the complex environment with 10% fetal bovine serum (FBS) and a serum sample. Meanwhile, the detection limit could be as low as 83.2 fM. In addition, this method effectively reduced the effect of photobleaching and maintained high stability, which was expected to achieve efficient and sensitive miRNA detection.