Nov 22, 2016 - Based on Tetrahedral DNA Nanostructures and Duplex-Specific ... (3D) DNA tetrahedron-structured probes (TSPs) to increase the probe ...
Research Article www.acsami.org
Ultrasensitive and Multiple Disease-Related MicroRNA Detection Based on Tetrahedral DNA Nanostructures and Duplex-Specific Nuclease-Assisted Signal Amplification Fang Xu,† Haifeng Dong,*,† Yu Cao,† Huiting Lu,‡ Xiangdan Meng,† Wenhao Dai,† Xueji Zhang,*,† Khalid Abdullah Al-Ghanim,§ and Shahid Mahboob⊥ †
Research Center for Bioengineering and Sensing Technology, School of Chemistry and Bioengineering, University of Science & Technology Beijing, Beijing 100083, P. R. China ‡ School of Space and Environment, Beihang University, Beijing 100191, P. R. China § Department of Zoology, College of Science, P.O. Box 2455, King Saud University, Riyadh 11451, Saudi Arabia ⊥ Department of Zoology, Government College University, Faisalabad, Pakistan S Supporting Information *
ABSTRACT: A highly sensitive and multiple microRNA (miRNA) detection method by combining three-dimensional (3D) DNA tetrahedron-structured probes (TSPs) to increase the probe reactivity and accessibility with duplex-specific nuclease (DSN) for signal amplification for sensitive miRNA detection was proposed. Briefly, 3D DNA TSPs labeled with different fluorescent dyes for specific target miRNA recognition were modified on a gold nanoparticle (GNP) surface to increase the reactivity and accessibility. Upon hybridization with a specific target, the TSPs immobilized on the GNP surface hybridized with the corresponding target miRNA to form DNA−RNA heteroduplexes, and the DSN can recognize the formed DNA−RNA heteroduplexes to hydrolyze the DNA in the heteroduplexes to produce a specific fluorescent signal corresponding to a specific miRNA, while the released target miRNA strands can initiate another cycle, resulting in a significant signal amplification for sensitive miRNA detection. Different targets can produce different fluorescent signals, leading to the development of a sensitive detection for multiple miRNAs in a homogeneous solution. Under optimized conditions, the proposed assay can simultaneously detect three different miRNAs in a homogeneous solution with a logarithmic linear range spanning 5 magnitudes (10−12−10−16) and achieving a limit of detection down to attomolar concentrations. Meanwhile, the proposed miRNA assay exhibited the capability of discriminating single bases (three bases mismatched miRNAs) and showed good eligibility in the analysis of miRNAs extracted from cell lysates and miRNAs in cell incubation media, which indicates its potential use in biomedical research and clinical analysis. KEYWORDS: multiple microRNA detection, duplex-specific nuclease, signal amplification, tetrahedral DNA nanostructures, ultrasensitive microRNA detection ogy among family members, small sizes, and low abundance.13 Traditional miRNA detection methods including microarrays,14 real-time polymerase chain reaction (RT-PCR),15 Northern blot,16 and in situ hybridization17 suffer from some instinctive inefficiencies such as poor sensitivity and expensive equipment, and they are time-consuming and require complex operation,18 which all limit their widespread applications. Highly sensitive, selective, and simple strategies are continuously developed for miRNA detection.19−21
1. INTRODUCTION MicroRNA (miRNA) is a class of endogenous regulatory noncode small molecules with lengths of 18−22 nucleotides1 that have attracted intense interest because of their pivotal roles in normal and pathologic processes.2−4 Importantly, the aberrant expression levels of human miRNAs are related to several diseases including cancer,5 hepatitis,6,7 malignancies,8 and neurodegeneration.9 Accumulated evidence has demonstrated that miRNA is a potential class of biomarker candidates for cancer classification,10 early disease diagnosis,11 and prognosis.12 However, the development of accurate, unbiased analysis of miRNA detection techniques for miRNA-based biomedical applications is still a major challenge because of the intrinsic characteristics of miRNA, including sequence homol© 2016 American Chemical Society
Received: September 27, 2016 Accepted: November 22, 2016 Published: November 22, 2016 33499
DOI: 10.1021/acsami.6b12214 ACS Appl. Mater. Interfaces 2016, 8, 33499−33505
Research Article
ACS Applied Materials & Interfaces
Scheme 1. Schematic Illustration of Multiple miRNA Detection Based on Tetrahedral DNA Nanostructures and DSN-Assisted Signal Amplificationa
a
The TSP probes immobilized on the GNP surface hybridize with specific targets to form DNA−RNA heterogeneous structures, and DSN recognizes the heterogeneous structures to cleave the DNA strand to produce the corresponding fluorescence, while the released miRNAs can trigger another isothermal amplification cycle for sensitive miRNA detection.
attomolar sensitivities (
