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.
Schematic of a fluorometric microRNA assay based on two-step amplification involving strand displacement replication and rolling circle amplification. DNA probe SYBR Green II is then bound to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA.
Xiaoya Ren,Xu Cui,Ying Zeng,Yimei Guo,Junhua Chen,Jiafeng Pan
In this research article, an intelligent clustered regularly interspaced short palindromic repeats (CRISPR)/Cas14a-based logic system was developed for microplastic monitoring on the basis of catalytic hairpin assembly (CHA) and DNA walkers on magnetic beads. Polyvinyl chloride (PVC) and polystyrene (PS) can activate the CHA-DNA walker sensing system to generate numerous single-stranded DNA fragments that can be recognized by Cas14a. After the cleavage reaction targeting the fluorophore-quencher reporter, a high Cy5 signal can be observed. The fluorescent biosensor is ultrasensitive, with a limit of detection of 3.4 ng/ml for PVC and 4.1 ng/ml for PS. Using PVC and PS as the two inputs, an OR logic gate was further developed. The input combinations of (10), (01), and (11) can give an output of 1. The sensing system is robust and can work in real water samples with good accuracy. Our proposed biosensor can provide an ultrasensitive and intelligent tool for microplastic monitoring.
危险品化学品经营许可证（带存储）