Rapid and ultrasensitive detection of microRNA by target-assisted isothermal exponential amplification coupled with poly (thymine)-templated fluorescent copper nanoparticles

Abstract

We devised a novel method for rapid and ultrasensitive detection of target microRNA (miRNA) by employing target-assisted isothermal exponential amplification (TAIEA) combined with poly (thymine)-templated fluorescent copper nanoparticles (CuNPs) as signaling probes. The target miRNA hybridizes to the unimolecular template DNA and works as a primer for the extension reaction to form double-stranded product, which consequently generates two nicking endonuclease recognition sites. By simultaneous nicking and displacement reactions, exponential amplification generates many poly (thymine) strands as final products, which are employed for the synthesis of fluorescent CuNPs. Based on the fluorescent signal from CuNPs, target miRNA is detected as low as 0.27 fM around 1 h of total analysis time. The diagnostic capability of this system has been successfully demonstrated by reliably detecting target miRNA from different cell lysates, showing its great potential towards real clinical applications.

Research advances in the detection of miRNA

“…[19] reported an ultrasensitive miRNA detection via isothermal exponential amplification reaction (EXPAR)-assisted AuNP amplification, which was inspired by the design of Park et al. [20]. Based on the method, they achieved the quantification of miRNAs in a linear range from 50 fM to 10 nM with a detection limit of 46 fM.…”

“…Traditional methods for miRNA detection include northern blotting [[10], [11], [12]], microarray analysis [13,14] and quantitative polymerase chain reaction (qPCR) [15,16]. For new technology methods, in order to improve the sensitivity and selectivity of miRNA detection, these methods always rely on signal amplification strategies, such as nanoparticle-based amplification [[17], [18], [19], [20], [21]], isothermal exponential amplification [[22], [23], [24]], rolling circle amplification (RCA) [[25], [26], [27]], hybridization chain reaction (HCR) [[28], [29], [30]] and combination of these amplification strategies [[31], [32], [33]]. Herein, we summarize some of them and discuss their advantages and limitations for improving miRNA detection design.…”

Ultrasensitive DNA detection based on target-triggered rolling circle amplification and fluorescent poly(thymine)-templated copper nanoparticles

“…26 Notably, poly T-CuNPs are known to be more sensitive and quickly prepared as compared to DNA-templated silver nanoclusters (DNA-AgNCs), a wellknown uorescent signal reporter for the nucleic acid detection. [27][28][29][30][31] In principle, the presence of target DNA induces the circularization of a padlock DNA probe composed of a target DNA-specic region and a poly(adenine) region, and thus the subsequent RCA is promoted to generate a long, concatemeric ssDNA with a lot of repetitive poly T sequences. As a result, a large number of highly uorescent poly T-CuNPs are produced.…”

An impedimetric determination of alkaline phosphatase activity based on the oxidation reaction mediated by Cu2+ bound to poly-thymine DNA

“…Recently, Qing et al described an ascorbic acid-catalyzed reduction of Cu 2+ entrapped within poly-thymine DNA probe for the formation of uorescence copper nanoparticle (CuNP), which has been used for development of various bioassays. [31][32][33][34][35][36][37][38] Taking this unique feature, Ocaña et al developed an impedimetric method for sensing Cu 2+ . 39 In this method, the rate of ascorbic acid oxidation was signicantly enhanced by DNAtemplated Cu 2+ on the electrode, 40,41 and consequently the electrode surface was covered with oxidation products, resulting in an increase in the electron transfer resistance (R et ) of the electrode.…”