Ultrasensitive and Multiple Disease-Related MicroRNA Detection

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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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12214 • Publication Date (Web): 22 Nov 2016 Downloaded from http://pubs.acs.org on November 24, 2016

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ACS Applied Materials & Interfaces

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, § 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.

KEYWORDS:

Multiple

microRNA

detection,

duplex-specific

nuclease,

amplification, tetrahedral DNA nanostructures, ultrasensitive microRNA detection.

ACS Paragon Plus Environment

signal


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ABSTRACT: A highly sensitive and multiple microRNA (miRNA) detection method by combining three-dimensional (3D) DNA tetrahedron-structured probes (TSP) for increase probe reactivity and accessibility with duplex-specific nuclease (DSN) for signal amplification for sensitive miRNA detection was proposed. Briefly, three-dimensional (3D) DNA TSPs labeled with different fluorescent dyes for specific target miRNAs recognition were modified on gold nanoparticle (GNP) surface to increase reactivity and accessibility. Up hybridization with specific target miRNA that the TSPs probes 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 structure to produce specific fluorescent signal corresponding to specific miRNA, while the released target miRNA strands can initiate another cycle resulting in a significant signal amplification for sensitive miRNAs detection. Different targets can produce different fluorescent signals, leading to the development of a sensitive detection for multiple miRNAs in homogeneous solution. Under optical conditions, the proposed assay can simultaneously detect three different miRNAs in homogeneous solution with a logarithmic linear range spanning 5 magnitudes (10-12-10-16) and achieve a limit of detection (LOD) down to attomolar concentrations. Meanwhile, the proposed miRNA assay exhibited capability to discriminate single-base, three-bases mismatched miRNAs and showed good eligibility in analysis miRNAs extracted from cell lysates and miRNAs in cell incubation media, which indicates its potential use in the biomedical research and clinical analysis.


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■

INTRODUCTION

MicroRNA (miRNA), a class of endogenous regulatory noncode small molecules with a length of 18-22 nucleotides,1 which have attracted intense interest due to 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 neuro-degeneration.9 Accumulated evidence has demonstrated that miRNA is a potential class of biomarker candidate for cancer classification,10 early disease diagnosis,11 and prognosis.12 However, development of accurate, unbiased analyzing miRNA detection techniques for miRNA-based biomedical applications is still a major challenge because of the intrinsic characteristics of miRNA, including sequence homology 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 blot16 and

in situ hybridization17 suffer from some instinctive inefficiencies such as poor sensitivity, expensive equipment, they are time-consuming and require complex operation,18 which limit their wide spread applications. Highly sensitive, selective and simple strategies are continuously developed for miRNA detection.19-21 Recently, various isothermal enzyme-assisted amplification strategies making a 1:1 hybridization event in association with numerous enzymes catalytic response have been intensely employed in nucleic acid biosensor, which have led to sharp detection signal amplification and improved sensitivity.22-24 Duplex-specific nuclease (DSN) is a highly stable endonuclease purified from hepatopancreas of Red King (Kamchatka)



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crab, which presents a stronger preference for cleaving DNA in DNA-RNA hybrids, attracting great interests for isothermal amplification cycle.25-27 Degliangeli and co-workers designed an absolute and direct miRNA detection method with a limit of detection (LOD) of 0.2 fM by using fluorescent dye labeled DNA strands modified PEGylated gold nanoparticles (GNPs) probes and DSN-assisted isothermal amplification.28 Ren et al developed a sensitive and selective electrochemical miRNAs biosensor based on miRNA strand triggered DSN cleavage of hybridized DNA capture probes, allowing detection of miRNA in serum down to 1.0 fM.26 In addition to signal amplification, probe immobilization is also a critical factor and should be considered. As for nucleic acid biosensor, traditional single-stranded or hairpin-based probe immobilization purports some inefficiencies such as the crowding of strand probes at high concentration or long strand length, and adhering along the surface at low concentrations or short strand length. It leads to reduced accessibility of the probe to the target, thus decrease the sensitivity of the biosensor.26,28 Three-dimensional (3D) DNA tetrahedron-structured probe (TSP) provides a nano-engineered interface for spatial control and enhanced probes accessibility arising from unparalleled self-recognition properties of DNA molecules on the surface, which shows great promise in nucleic acid biosensors.29-32 Fan and co-workers immobilized 3D DNA TSP modified with sulfur at three vertices on gold electrode surfaces through Au-S interaction to develop a DNA-based biosensor platform for DNA detection resulting improved sensing ability due to enhanced spatial positioning range and enhanced probe accessibility on the surface.33 The same group further designed


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an ultrasensitive electrochemical miRNA biosensor with LOD down to attomolar sensitivities (

Ultrasensitive and Multiple Disease-Related MicroRNA Detection

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