A DNA tetrahedral structure-mediated ultrasensitive fluorescent microarray platform for nucleic acid test

https://doi.org/10.1016/j.snb.2020.128538Get rights and content

Abstract

Microarrays are key platforms for biomolecule detection owing to their parallelizability and amenability for high-throughput. However, conventional fluorescent microarrays still suffer from low specificity and sensitivity, therefore are unsuitable for detection of low abundance nucleic acids. In this study, we demonstrate a universal microarray platform with high specificity and ultra-sensitivity for fluorescent detection of DNA and micro RNA (miRNA), which employs a DNA tetrahedral structured probe (DTSP) together with a hybridization chain reaction (HCR) based signal amplification technique. By precisely modulating the number of base pairs on each tetrahedron side, we developed three different sized DTSPs: 17 base pairs, 26 base pairs, and 37 base pairs (DTSP-17, DTSP-26 and DTSP-37, respectively). A low detection limit of 10 aM was obtained by DTSP-26, notably much lower than detection limits of prior methods. Furthermore, our microarray platform can distinguish single base DNA mismatches and thus exhibits single nucleotide specificity. Lastly, our microarray platform can be implemented for miRNA detection, as we demonstrate in a mimic medium, demonstrating potential for its use in clinical diagnosis.

Introduction

In recent years, many techniques and methods have been reported for nucleic acid detection and testing, including nanopore sensors [1], nanomaterial based optical sensors [[2], [3], [4], [5], [6], [7]], colorimetric sensors [8], northern blotting [9], quantitative polymerase chain reaction (qPCR) [10,11] and sequencing [12]. Microarray technologies are regarded as a promising method in life science research and clinical diagnostics, and are also widely used for nucleic acids detection [[13], [14], [15]] because of their ability to parallelize testing in a high-throughput manner. Different strategies have been developed for improving detection performance of microarray analysis. For example, Quantum dots (QDs) with high fluorescence quantum yields and long lifetimes have been introduced to increase signals [14]. Three-dimensional dextran-coated microarrays coated have also been designed to decrease background signals for the purpose of increasing sensitivity [16]. However, the sensitivity, selectivity, and reproducibility of microarrays remain insufficient for low abundant micro RNA (miRNA) detection [17]. Aforementioned limitations in microarray technologies are due to 1) the softness and easy to fall down of many microarray probes, which can lower the hybridization efficiency, and 2) high probe loading densities needed for the detection of low abundance analytes that often decrease the microarray assay selectivity. Hence, microarray technologies for low-abundance nucleic acid detection is an area requiring new strategies for programmable molecular recognition.

DNA nanotechnology has recently attracted much interest due to its nanoscale programmability and inherent biocompatibility [[18], [19], [20], [21], [22]]. DNA molecules can be self-assembled into DNA nanostructures [23] with various sizes, shapes, and geometries for applications in protein structure determination, medical diagnosis, biosensing, and drug delivery [[24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36]]. Among the many DNA nanostructure form factors is the Tetrahedron DNA nanostructures (TDNs), which was first designed by Goodman et al. [37] and initially used as a probe for biosensing by Pei et al. [28]. Many biosensing platforms have since been established for bioanalysis of DNA, miRNA, protein, and cells, based on DNA tetrahedra [31,[38], [39], [40]]. The advantages of the DNA tetrahedron for molecular detection lies in two aspects: first, the rigid tetrahedral scaffold can fix probes in a straightforward direction. Second, the controllable size can regulate the distance between probes to reduce hybridization steric hindrance [38,[40], [41], [42], [43]]. By programming the size of the DNA tetrahedral structured probe, we can control its surface adsorption properties [44] and improve the effectiveness of probe recognition by optimizing ligand-probe binding. An exemplary study by Li et al. [40] demonstrated that DNA tetrahedron-based microarrays can achieve a 10 fM limit of detection (LOD) for a target miRNA, however, this LOD remains below the ∼aM target sensitivity needed for detection of low abundance nucleic acids tests.

Separately, the hybridization chain reaction (HCR) involves the triggering of two stable DNA hairpins by an initiator to form long double helix strings through a cascade hybridization reaction. Labeled DNA can act as an amplifying tool for biodetection and bioimaging applications [16,31,39,[45], [46], [47]] whereby the HCR reaction proceeds to magnify the signal and can significantly increase the sensitivity of nucleotide detection. This amplification method is an enzyme-free reaction that can react at room temperature, and has been shown to achieve a 10 fM LOD [16] when incorporated into a microarray platform. HCR is an attractive platform for signal amplification because it is not an enzyme-coupled method, enabling its implementation in a broad range of pH, temperature, and buffer media conditions [48].

Given the orthogonal advantages of DNA nanotechnology and HCR, herein we developed a platform combining both DNA tetrahedral structured-probes (DTSP) and HCR to produce a microarray platform with aM sensitivity. Firstly, we designed three differently sized DTSPs (DTSP-17, DTSP-26, DTSP-37). Secondly, we employed HCR to increase the sensitivity of our microarray platform by introducing coupling repeat units on the DTSP with fluorescent dyes. Our combined DSTP and HCR based microarray platform achieved a 10 aM LOD for a target low-abundance DNA, and enabled detection of a single-base mismatch. We lastly demonstrated that our microarray platform can be used for miRNA detection in simulated serum, suggesting our platform can be useful in biodetection and clinical assays for low-abundance nucleic acids.

Section snippets

Design of the DTSP−HCR microarray platform

To address limitations in microarray sensitivity and specificity, herein we combine DSTP and HCR to maximize the performance of microarray technology. In our design, the DTSP contains one ssDNA extension as the detection probe at one vertex, and three amino groups at the remaining three vertices for tetrahedron immobilization via covalent coupling between amino groups on ssDNA and aldehyde groups to form imine bond on the glass surface (Fig. 1). With this approach, the DTSP anchored to the

Conclusions

We present an ultrasensitive microarray platform mediated by programmable DNA nanotechnology, employing DNA tetrahedral structure as probes together with HCR signal amplification technology. Compared to conventional sandwich assays that are implemented to detect DNA and RNA targets, our DTSP−HCR strategy exhibited over 10-fold increased fluorescence signals towards target polynucleotides, which enabled a lower ∼aM limit of detection than previously reported for microarray technology. We

Sources of materials

All chemicals were purchased from Sangon Biotech (Shanghai, China) Co., Ltd. unless otherwise stated. Aldehyde-functionalized slides were purchased from Capital Bio Corp. (Beijing, China). Dimethyl sulfoxide(DMSO) was purchased from Sigma-Aldrich (St. Louis, Mo). Fetal calf serum was purchased from Invitrogen (Shanghai, China). All oligonucleotides were synthesized and purified by Sangon Biotech Co. Ltd (Shanghai, China), and the sequences are listed in Table S1. The mature miRNAs were

Author contributions

X.Q.M. supervised the whole project and guided writing the manuscript. H.Z. and X.X.L. carried out the experiments, analyzed the data and wrote the manuscript. Y.Z, M.L and L.H.W verified the analytical methods and supervised the findings of this work. X.L.L helped with the spotting experiments. All other authors discussed the results and contributed to the final manuscript.

Notes

The authors have declared that no competing interest exists.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We acknowledge the support from National Natural Science Foundation of China (21605153), National Key Research and Development Program of China (2016YFC0100600), National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2015BAI02B02), Science and Technology Service Network Initiative, CAS (KFJ-EW-STS-140, KFJ-EW-STS096), and Shanghai Municipal Science and Technology Commission (15441905000, 16DZ1930700).

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