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A novel electrochemiluminescence (ECL) biosensor was fabricated for miRNA-162a detection by using silver nanoclusters/molybdenum disulfide (AgNCs@MoS2) as an ECL material, peroxodisulfate (S2O82−) as a co-reactant, and semicarbazide (Sem) as a co-reaction accelerator. Firstly, hairpin probe Ha modified on AgNCs@MoS2/GCE was unfolded based on its hybridization with target microRNA. Then, the unfolded Ha can further be hybridized with another hairpin DNA of Hb on (AuNPs-semicarbazide)@Cu-MOF, resulting in the release of target microRNA, which further causes a cyclic hybridization. This creates more (AuNPs-semicarbazide)@Cu-MOF on the electrode surface, achieving cyclic hybridization signal amplification. Strikingly, due to the presence of Sem, accelerating the reduction of S2O82− and resulting in the generation of more oxidant intermediates of SO42−, the amount of excited states of Agincreases to further amplify the ECL signal. The biosensor exhibited high sensitivity with a low LOD of 1.067 fM, indicating that the introduction of co-reaction accelerators can provide an effective method for signal amplification. The applicability of this method was assessed by investigating the effect of Pb(II) ion on miRNA-162a expression level in maize seedling leaves. [ABSTRACT FROM AUTHOR]

The authors describe a fluorometric assay for microRNA. It is based on two-step amplification involving (a) strand displacement replication and (b) rolling circle amplification. The strand displacement amplification system is making use of template DNA (containing a sequence that is complementary to microRNA-21) and nicking enzyme sites. After hybridization, the microRNA strand becomes extended by DNA polymerase chain reaction and then cleaved by the nicking enzyme. The DNA thus produced acts as a primer in rolling circle amplification. Then, the DNA probe SYBR Green II is added to bind to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA. The method has a wide detection range that covers the10 f. to 0.1 nM microRNA concentration range and has a detection limit as low as 1.0 fM. The method was successfully applied to the determination of microRNA-21 in the serum of healthy and breast cancer patients. [ABSTRACT FROM AUTHOR]

A photoelectrochemical biosensor is described for sensitive detection of microRNA-162a. A multiple amplification strategy is employed that involves (a) isothermal strand displacement polymerase reaction; (b) terminal deoxynucleotidyl transferase-mediated extension, (c) amplification of streptavidin-coated gold nanoparticles, and (d) biotin functionalized alkaline phosphatase. Graphite-like C3N4 (g-C3N4) nanosheets were used as photoactive material. By using these amplification strategies, the detection limit is as low as 0.18 fM of microRNA, and the photocurrent increases linearly with the concentration of microRNA-162a in the range from 0.5 fM to 1 pM. The method was successfully applied to quantify the expression level of microRNA-162a in total RNA extracted from the leaves of maize seedlings after incubation with the chemical mutagen ethyl methanesulfonate. The results confirmed the applicability of the method to the analysis of practical biological samples.Schematic of a photoelectrochemical microRNA assay based on a multiple amplification strategy involving (a) isothermal strand displacement polymerase reaction; (b) terminal deoxynucleotidyl transferase-mediated extension, (c) amplification of streptavidin-coated gold nanoparticles, and (d) biotin functionalized alkaline phosphatase. [ABSTRACT FROM AUTHOR]