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MicroRNAs play crucial role in post-transcriptional regulation for gene expression in animals, plants, and viruses. For the better understanding of microRNA and its functions, it is very important to develop effectively analytical method for microRNA detection. Herein, a novel electrochemical biosensor was fabricated for sensitive and selective detection of microRNA based on poly(U) polymerase mediated isothermal signal amplification, where poly(U) polymerase can catalyze the template independent addition of UMP from UTP to the 3' end of RNA. Using this activity, the target microRNA can be successfully labeled with biotin conjugated UMPs at its 3'-end using biotin conjugated UTP (biotin-UTP) as donor. Then, the avidin conjugated alkaline phosphatase can be further captured to the 3'-end of the target microRNA based on the specific interaction between biotin and avidin. Finally, under the catalytic activity of alkaline phosphatase, the substrate of p-nitrophenyl phosphate disodium salt hexahydrate can be hydrolyzed to produce 4-nitrophenol. According to the relationship between the electrochemical signal of p-nitrophenol and the concentration of microRNA-319a, the content of microRNA-319a can be detected. This signal amplification method is simple and sensitive. The developed method can detect as low as 1.7 fM microRNA and produce precise and accurate linear dynamic range from 10 to 1000 fM. The fabricated biosensor was further applied to detect the expression level change of microRNA-319a in rice seedlings after incubation with five kinds of different phytohormones., (Copyright © 2015 Elsevier B.V. All rights reserved.)

MicroRNA (miRNA) is riveting nowadays due to its close relevance to human malignancies. Exploiting a fast and convenient biosensor for sensitive and specific detecting miRNA is necessary and it has significant meaning for oncology. In this work, to understand the relationship between miRNA-21 and gastric cancer, we employ T4 RNA ligase 2 to initiate specific ligation reaction depending on the sequence of target RNA, and T7 exonuclease to catalyze the first stage cyclic enzymatic amplification method (CEAM) specifically, which also resting on the target RNA sequence, and the second stage CEAM to further amplify the response resulting from the initial target RNA. Our two-stage CEAM strategy can detect miRNA-21 as low as 0.36fM with remarkable specificity, and most importantly, it can be used to inspect the expression level of miRNA-21 in blood serum of gastric cancer patients. This will offer new perspective for figuring out the pathways of miRNA-21 involving in cancer., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Herein, an electrochemical biosensor was fabricated for DNA demethylase detection based on DNA demethylation triggered endonuclease BstUI and Exonuclease III digestion. After the double-strand DNA was demethylated, it can be further digested by BstUI and formed a blunt end at the electrode surface. Then, the remained fragment of DNA-DNA duplex was further cleaved by exonuclease III and led to increased electrochemical signal. Based on this detection strategy, the biosensor showed high sensitivity with low detection limit of 0.15ng/mL. Moreover, the developed method also presented high selectivity and acceptable reproducibility. This work provides a novel detection platform for DNA demethylase detection., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A novel electrochemical method is developed for detection of DNA demethylation and assay of DNA demethylase activity. This method is constructed by hybridizing the probe with biotin tagged hemi-methylated complementary DNA and further capturing streptavidin tagged alkaline phosphatase (SA-ALP) to catalyze the hydrolysis reaction of p-nitrophenyl phosphate. The hydrolysate of p-nitrophenol (PNP) is then used as electrochemical probe for detecting DNA demethylation and assaying the activity of DNA demethylase. Demethylation of target DNA initiates a degradation reaction of the double-stranded DNA (dsDNA) by restriction endonuclease of BstUI. It makes the failed immobilization of ALP, resulting in a decreased electrochemical oxidation signal of PNP. Through the change of this electrochemical signal, the DNA demethylation is identified and the activity of DNA demethylase is analyzed with low detection limit of 1.3 ng mL(-1). This method shows the advantages of simple operation, cheap and miniaturized instrument, high selectivity. Thus, it provides a useful platform for detecting DNA demethylation, analyzing demethylase activity and screening inhibited drug., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Taking advantage of the special exodeoxyribonuclease activity of T7 exonuclease, a simple, sensitive, selective, and label-free microRNA biosensor based on the cyclic enzymatic amplification method (CEAM) has been proposed. First, thiol functionalized DNA probes were assembled onto a gold nanoparticles modified gold electrode surface through a Au-S bond, followed by hybridizing with target miRNA. Subsequently, DNA in RNA/DNA duplexes was digested by T7 exonuclease, which can release the microRNA molecules from the electrode surface and return into the buffer solution. Meanwhile, the released microRNA can further hybridize with the unhybridized DNA probes on the modified electrode surface. On the basis of it, an isothermal amplification cycle is realized. The T7 exonuclease-assisted CEAM achieved a low detection limit of 0.17 fM. Moreover, this assay presents excellent specificity with discriminating only a single-base mismatched microRNA sequence. Furthermore, this work can also be applied to detect avian leukemia based on the decreased expression level of microRNA-21.