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

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.