| MicroRNAs(MiRNAs) are a kind of short endogenousnonprotein coding RNA, which play a critical role in gene expression of most eukaryotic organisms from plants to animals.More and more evidence has demonstarted that abnormal repression of miRNA is associated with many diseases, including diabetes, cancer, and stroke induced tissue injury. However,miRNA detection is also challenged from their short lengths, low abundance, susceptibility to degradation, and sequence similarity among family members. The current widely used standard methods for miRNA analysis are Northern blotting, real-time PCR, and microarrays.Northern blotting is viewed as a standard assay method for miRNAs detection, but it is a time- and sample-consuming and semiquantitative detection method with low sensitivity and throughput. Without an elaborate separation and enrichment process, it is difficult to achieve sufficient sensitivity in biological sample analysis. The real-time PCR(RT-PCR) approach shows high detection sensitivity(about 100 fM) and good specificity, but it generally requires complex and tedious steps for miRNAs isolation and purification. It also needs to reverse transcription to cDNA prior to the amplification step. Furthermore, the apparatus for RT-PCR is very expensive. Analysis techniques based on microarray make multiple miRNA analysis feasible, but it frequently suffers from poor reproducibility and inaccuracy, currently resulting from cross hybridization and nonspecific adsorption. Thus, the development of fast, specific,sensitive, and convenient methods for miRNA analysis is urgently needed.(1) In this work, a photoelectrochemical(PEC) biosensor was fabricated for sensitive and specific detection of miRNA based on Bi2S3 nanorods and enzymatic signal amplification.Using the catalytic effect of alkaline phosphatase on L-ascorbic acid 2-phosphate trisodium salt(AAP), ascorbic acid(AA) was in situ generated and used as electron donor. Based on this, a signal-on protocol was successively achieved for miRNAs detection due to the dependence of photocurrent response on the concentration of electron donor of AA. The results demonstrated that the photocurrent response enhanced with increasing the hybridized concentration of miRNA. Under the amplification of the immunogold labeled streptavidin(SA-AuNPs), a low detection limit of 1.67 fM was obtained. The fabricated biosensor showed good detection stability and specificity, and it could discriminate only one-base mismatchedmiRNA sequence. Moreover, the down-regulated expression of miRNA-21 in DF-1 chicken fibroblast cells infected with subgroup J avian leukemia virus(ALVs) was confirmed by the developed method, indicating that miRNA-21 might be a new biomarker for avian leukemia.This work opens a different perspective for miRNAs detection and early diagnose of avian leukemia.(2) Taking advantage of the special exodeoxyribonuclease activity of T7 exonuclease, a simple, sensitive, selective, and label-free miRNA 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 miRNA molecules from the electrode surface and return into the buffer solution. Meanwhile, the released mi RNA 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 miRNA sequence. Furthermore,this work can also be applied to detect avian leukemia based on the decreased expression level of miRNA-21.(3) In this work, we proposed a selective and sensitive biosensor for miRNA detection based on the high specificity and affinity of anti-DNA:RNA hybrids antibody(S9.6 antibody)and alkaline phosphatase catalytic signal amplification. Briefly, after the hybridization of probe DNA and the target miRNA, the S9.6 antibody can be captured on the electrode surface through antigen-antibody immunoreaction. Then, alkaline phosphatase labeled goat anti-mouse IgG(ALP-IgG) was further captured on the electrode surface through the specific recognition effect between the primary antibody and the secondary antibody. Finally, ALP catalyzed the hydrolysis reaction of p-nitrophenyl phosphate to generate p-nitrophenol,resulting in a electrochemical oxidation signal. The simple signal amplification assay performed a successful linear range from 0.5-500 fM with a detection limit of 0.40 fM.Moreover, this biosensor exhibited high selectivity with discriminating only single-base mismatched miRNA sequence. Additionally, the simplicity of this method hold a greatpromise for further investigation the role of miRNAs in phytohormone signaling transduction.(4) We demonstrate a photo-electrochemical biosensor for the sensitive and specific detection of mi RNA using Bi2S3 nanorods as a photoactive material and streptavidin as the unit that inhibits photocurrent. Bi2S3 nanorods were synthesized hydrothermally in organic phase and displayed excellent light-to-current conversion efficiency. The Bi2S3 was deposited on an indium tin oxide(ITO) slice and then modified with gold nanoparticles onto which biotinylated hairpin probe DNA was deposited as a monolayer. Following hybridization between the biotinylated probe DNA and the target mi RNA, the stem-loop structure of the probe DNA was unfolded and the biotin directed outwards into the solution. Streptavidin was then added to bind to biotin via the strong streptavidin-biotin interactions. This causes the photocurrent of the modified ITO to decrease due to steric hindrance that blocks the transfer of electrons from added ascorbic acid to the surface of the electrode. The method has a detection limit as low as 3.5 fM of miRNA and can excellently discriminate even singly mismatched miRNA. The method was successfully applied to investigate the effect of abscisic acid on the expression level of miRNA-159 a in seeds of Arabidopsis thaliana. We conclude that the assay presented here has a large potential as a method for quantification of miRNA and for studying the epigenetic regulation of flowering plants.(5) MiRNAs(miRNAs) regulate a wide range of biological processes, and their abnormal expressions are related to the growth and development of plants. Thus, a simple,rapid, and highly sensitive assay for miRNA detection is of great significance. In this work, a label-free and ultrasensitive assay for miRNA detection using protein cage nanoparticles has been developed. Apoferritin-encapsulated Cu nanoparticles(Cu-apoferritin) could be immobilized on the electrode through special reaction between amino and carboxyl.Hybridization event between the probe DNA and the target miRNA-159 a is confirmed by electrochemical oxidation signal after Cu released into the detection buffer by adjusting the pH. This assay is highly selective and sensitive with a low detection limit of 3.5 fM.Moreover, the developed method can even discriminate single-base mismatched strand between the complementary targets. The effect of abscisic acid on the expression level of miRNA-159 a in Arabidopsis thaliana seeds was also investigated. |
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