Abstract
MicroRNAs (miRNAs) have emerged as the promising molecular biomarkers for early diagnosis and enhanced understanding of the molecular pathogenesis of cancers as well as certain diseases. Here, a facile, label-free, and amplification-free electrochemical biosensor was developed to detect miRNA by using DNA origami nanostructure-supported DNA probes, with methylene blue (MB) serving as the hybridization redox indicator, for the first time. Specifically, the use of cross-shaped DNA origami nanostructures containing multiple single-stranded DNA probes at preselected locations on each DNA nanostructure could increase the accessibility and the recognition efficiency of the probes (due to the rational controlled density of DNA probes). The successful immobilization of DNA origami probes and their hybridization with targeted miRNA-21 molecules was confirmed by electrochemical impedance spectroscopy and cyclic voltammetry methods. A differential pulse voltammetry technique was employed to record the oxidation peak current of MB before and after target hybridization. The linear detection range of this biosensor was from 0.1 pM to 10.0 nM, with a lower detection limit of 79.8 fM. The selectivity of the miRNA biosensor was also studied by observing the discrimination ability of single-base mismatched sequences. Because of the larger surface area and unprecedented customizability of DNA origami nanostructures, this strategy demonstrated great potential for sensitive, selective, and label-free determination of miRNA for translational biomedical research and clinical applications.
Highlights
MicroRNAs, a group of short noncoding RNAs (∼19−23 nucleotide long), play a vital role in cell growth, gene expression, and apoptosis.[1]
Pei et al demonstrated the use of a DNA tetrahedral probe, combined with varied amplification strategies, to detect miRNAs with high sensitivity and specificity.[23−33] In this design, DNA tetrahedral nanostructures were immobilized on the electrode surface, via the strong Au−S interaction between three thiol-modified vertices and the gold surface
When the hybrids of the DNA probe−miRNA were formed, the methylene blue (MB) molecules were bound to the hybridization chain, and the corresponding oxidation peak current of MB was directly proportional to the miRNA concentration
Summary
MicroRNAs (miRNAs), a group of short noncoding RNAs (∼19−23 nucleotide long), play a vital role in cell growth, gene expression, and apoptosis.[1]. Pei et al demonstrated the use of a DNA tetrahedral probe, combined with varied amplification strategies (such as redox enzyme incorporation and hybridization chain reaction), to detect miRNAs with high sensitivity and specificity.[23−33] In this design, DNA tetrahedral nanostructures were immobilized on the electrode surface, via the strong Au−S interaction between three thiol-modified vertices and the gold surface. The immobilization of DNA origami probes on a working electrode (WE) surface was realized, via electrostatic adsorption between a cationic and biodegradable chitosan film[37,38] and negatively charged DNA origami nanostructures This method offers the advantage of providing a simple yet stable attachment of DNA origami on an electrode surface because of the large contact surface area of the DNA origami, and avoiding the use of modified thiol-DNA or biotin-DNA. With the facile fabrication method that does not require laborious labeling, probe immobilization, and signal amplification, our strategy promises the aforementioned advantages in determining the concentration of miRNA
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