Fluorescent Biosensor Based On Nucleic Acid Amplification And Its Application In MicroRNA Detection

In this paper,the function and detection methods of miRNA,nucleic acid isothermal amplification and fluorescence spectroscopy were introduced in detail and two ultra-sensitive methods for miRNA analysis were proposed.In our experimental program1,we constructed an ultra-sensitive fluorescent biosensor for miRNA-155 detection based on branched rolling circle amplification(RCA)combining with Nb.BbvCI.In the presence of the target miRNA-155,it first initiates the backbone rolling circle amplification after opening the hairpin probe 1and the reaction product initiates a branched rolling circle amplification after opening the hairpin probe 2.After the molecular beacon is opened by the product of branched rolling circle amplification,the Nb.BbvCI site is formed,which in turn initiates the cyclic cleavage reaction of Nb.BbvCI to the molecular beacon,resulting in a large number of molecular beacons being cut off and greatly enhanced fluorescence signal strength and detection sensitivity.In our experimental program2,we constructed a highly sensitive,specific and simple fluorescent biosensor to achieve ultra-sensitive analysis of miRNA-122.In the presence of the target miRNA-122,it first combines with the 5'end of the hairpin probe,and then the 3' end of help probe combines with the 3' end of hairpin probe.Under the action of phi29 DNA polymerase and Nb.BbvCI,the recycling of target miRNA and the formation and reuse of secondary targets were realized,and a great number of primers were generated.At this time,the primer can bind to the circular template DNA and initiate rolling circle amplification and produce a long singlestranded DNA with a repeating sequence complementary with MB.At the same time,MBs were opened by the rolling circle amplification product and formed Nb.BbvCI cleavage sites,therefore Nb.BbvCI can carry out cyclic cutting reaction for MB,which greatly enhances the fluorescent signal intensity.This strategy we proposed can quantitatively detect miRNA-122 in a dynamic range from 10 aM to 10 pM with a detection limit as low as 3.9 aM.