Ratiometric Fluorescent Biosensor Based on Self-Assembled Fluorescent Gold Nanoparticles and Duplex-Specific Nuclease-Assisted Signal Amplification for Sensitive Detection of Exosomal miRNA

Abstract

The sensitive detection of cancer-associated exosomal microRNAs shows enormous potential in cancer diagnosis. Herein, a ratiometric fluorescent biosensor based on self-assembled fluorescent gold nanoparticles (Au NPs) and duplex-specific nuclease (DSN)-assisted signal amplification was fabricated for sensitive detection of colorectal cancer (CRC)-associated exosomal miR-92a-3p. In this biosensing system, the hairpin DNA modified with sulfhydryl and fluorescent dye Atto-425 at both ends is conjugated to fluorescent Au NPs through Au–S bonds, resulting in the quenching of Atto-425. The miR-92a-3p can open the hairpin of DNA and forms an miR-92a-3p/DNA heteroduplex, triggering the specific cleavage of DSN for the DNA in the heteroduplex. As a result, Atto-425 leaves the fluorescent Au NPs and recovers the fluorescence emission. The released miR-92a-3p can hybridize with another hairpin DNA and lead to a stronger fluorescence recovery of Atto-425 to form a signal amplification cycle. The stable fluorescence of Au NPs and the changing fluorescence of Atto-425 constitute a ratiometric fluorescent system reflecting the concentration of miR-92a-3p. This biosensor exhibits excellent specificity and can distinguish CRC patients from healthy individuals by detecting miR-92a-3p extracted from clinical exosome samples, showing the potential in CRC diagnosis.

Identification and detection of microRNA kidney disease biomarkers in liquid biopsies

“…The hairpin opened in the presence of the miRNA to form the hybrid, and then exogenous, duplex-specific nuclease enzyme was added to degrade the DNA strand of the hybrid RNA-DNA duplex and release the miRNA to react with further DNA hairpins. This strategy used a simpler enzyme-based catalytic cycle, resulting in an improved limit of detection of 45 fM, and the authors also demonstrated miR-92a upregulation in serum exosomederived samples from colorectal cancer patients [37].…”

“…Sun et al [37] employed a similar strategy to detect miR-92a, a biomarker of colorectal cancer, in serum exosomes. This method also involved an amplification cycle, in this case hairpin DNA probes labelled with an ATTO-425 fluorophore were attached to gold nanoparticles.…”

Gold Nanoparticles as a Biosensor for Cancer Biomarker Determination

“…A specific antibody with a label can identify the cleaved structure and interpret the structure using methods such as electrochemistry [112]. It has been shown that this duplex-specific nuclease approach can be used in conjunction with fluorescence [113,114], surface plasmon resonance [115], or SERS [116] for the identification of signals in other ways.…”

MicroRNA-nanoparticles against cancer: Opportunities and challenges for personalized medicine

“…This tool represents a potential innovative and specific system for CRC diagnosis. 139 Prussian blue NP (PBNP)-based fluorescent nanoprobes represent another type of biosensor for the diagnosis of CRC. PBNPs exploit the quenching ability to fluorophores to evaluate miR-92a and miR-21 levels, crucial CRC biomarkers.…”