Electrochemical Sensing of Exosomal MicroRNA Based on Hybridization Chain Reaction Signal Amplification with Reduced False-Positive Signals

Abstract

MicroRNAs (miRNAs) in cancer cell-derived exosomes are important cancer biomarkers. Herein, a sensitive hybridization chain reaction (HCR) electrochemical assay was fabricated for the detection of exosomal microRNA-122 (miR-122). The hairpin DNA (hpDNA) probes were first immobilized on the surface of a gold electrode. In the presence of miR-122, the hairpin structure of the hpDNA could be opened and triggered the HCR through the cross-opening and hybridization of two helper DNA hairpins. Long nicked double helixes generated from HCR are used to capture more RuHex and increase the signal of differential pulse voltammetry (DPV). In this assay, the density of the hpDNA probes on the surface of the gold electrode was precisely controlled by the simultaneous immobilization of hpDNA and short 12 nucleotides single-stranded DNA (S-12), providing a very high amplification efficiency. More importantly, the false positive signal could be reduced or completely eliminated by applying exonuclease I (Exo I) before the introduction of target miR-122. Under optimal conditions, the assay offers very high sensitivity with an attomolar level detection limit, a linear range with 9 orders of magnitude, and specificity in single mismatch discrimination. This sensitive electrochemical assay could successfully evaluate the miR-122 concentration in different cancer-derived exosomes, indicating its potential use in cancer diagnostics.

Applications of Bionano Sensor for Extracellular Vesicles Analysis

“…This simple DNA structural transformation could detect exosomal miR-21 with a LOD as low as 2.3 fM. Guo et al showed a sensitive electrochemical assay of miR-122 by HCR of the hairpin structure of the capture DNA on the surface of the electrode [91]. The miR-122 opened the hairpin DNA and triggered the HCR through the cross-opening and hybridization of two helper DNA hairpins.…”

MicroRNAs from Liquid Biopsy Derived Extracellular Vesicles: Recent Advances in Detection and Characterization Methods

“…According to the authors, this method is highly sensitive, cheap, and results are reproducible. However, their assay was not used yet to analyze patient samples [149].…”

Cascade Toehold-Mediated Strand Displacement Reaction for Ultrasensitive Detection of Exosomal MicroRNA

“…Boriachek et al 26 prepared DNA-modified magnetic beads for the capture and amplification-free measurement of the target. Guo et al 27 achieved DNA elongation on the electrode interface by hybridization chain reaction and determined exosomal miRNA levels with reduced false-positive signals.…”

“…Compared with some other nonenzymatic amplifications, like the hybridization chain reaction, the distance between electrochemical species and electrode is not only closer but also fixed, which is more suitable for quantitative analysis. 27…”