Hybridization Chain Reaction Amplification of MicroRNA Detection with a Tetrahedral DNA Nanostructure-Based Electrochemical Biosensor

Abstract

There remains a great challenge in the sensitive detection of microRNA because of the short length and low abundance of microRNAs in cells. Here, we have demonstrated an ultrasensitive detection platform for microRNA by combining the tetrahedral DNA nanostructure probes and hybridization chain reaction (HCR) amplification. The detection limits for DNA and microRNA are 100 aM and 10 aM (corresponding to 600 microRNAs in a 100 μL sample), respectively. Compared to the widely used supersandwich amplification, the detection limits are improved by 3 orders of magnitude. The uncontrolled surface immobilization and consumption of target molecules that limit the amplification efficiency of supersandwich are eliminated in our platform. Taking advantage of DNA nanotechnology, we employed three-dimensional tetrahedral DNA nanostructure as the scaffold to immobilize DNA recognition probes to increase the reactivity and accessibility, while DNA nanowire tentacles are used for efficient signal amplification by capturing multiple catalytic enzymes in a highly ordered way. The synergetic effect of DNA tetrahedron and nanowire tentacles have proven to greatly improve sensitivity for both DNA and microRNA detection.

Ultrasensitive electrochemical detection of avian influenza A (H7N9) virus DNA based on isothermal exponential amplification coupled with hybridization chain reaction of DNAzyme nanowires

“…EXPAR has the intrinsic merits of isothermal nature, high amplification efficiency and rapid amplification kinetics, which can provide 10 6 -10 9 fold amplification within minutes under isothermal conditions. In addition, metal nanoparticles (Sharifi et al, 2014;Wu et al, 2014), quantum dots (Zhu et al, 2014), DNA biobarcode (Xu et al, 2012;Zhou et al, 2014b) and labeled enzymes (Ge et al, 2014) were also the common examples of amplification strategy. However, the multiple assay steps, the addition of many exogenous reagents and the labeling process made them laborious and costly.…”

Enhanced electrochemical recognition of double-stranded DNA by using hybridization chain reaction and positively charged gold nanoparticles

“…Unlike other amplification approaches, HCR owns significant advantages including enzyme free, low background and PCR-like sensitivity (Ge et al, 2014;Huang et al, 2011). These characteristics make HCR be a promising candidate for the detection of DNA Chen et al, 2012;Niu et al, 2010;Bi et al, 2013;Li et al, 2014;Qian et al, 2015;Ren et al, 2015), protein (Song et al, 2014;Zhou et al, 2012;Dai et al, 2012;Zhang et al, 2012), metal ions Yuan et al, 2011;, cell (Choi et al, 2011) and small molecules by using electrochemical, fluorescent, colorimetric methods and so on.…”

Electrochemical Detection of RNA

“…Furthermore, the authors demonstrated the application of this sensor for analyzing miRNA expression levels in clinical samples from esophageal squamous cell carcinoma patients. An identical limit of detection (10 aM) was achieved by combining the use of tetrahedral DNA nanostructure probes as described above and hybridization chain reaction amplification within a total analysis time of more than 4 h. However, in this study, the authors showed no application of the biosensor with real samples (Ge et al 2014 probe (UIP) and two DNA adaptor strands which cooperatively hybridize to complementary parts of miRNA and the immobilized UIP. In the absence of target miRNA, the two adaptor strands cannot interact with the UIP due to the stable stemloop structure of the UIP which is thermodynamically more stable than the assembly with the adaptor strands.…”