Based On Restriction Endonuclease Amplified MiRNA Detection Biosensor

Aberrant expression of micro RNA(miRNA) is associated with development of cancers and diseases, so miRNA has become a tissue-based biomarker for cancer prognosis and diagnosis. Therefore, the novel methods for miRNA detection has become an irrespensible problem. Recently, on the basis of nicking endonuclease and polymerase, the autonomous polymerization-nicking isothermal synergetic enzyme amplification processes has been widely used for miRNA. Nonlinear amplification has higher efficiency and EXPAR is promising nonlinear amplification strategy of high sensitivity, low cost and suited for miRNAs real-time detection in the clinical samples. With high amplification efficiency,EXPAR can rapidly amplify short oligonucleotides within short time. Recently, based on enzymatic reaction used EXPAR, we designed two approach to finish the detection. First, a novel trigger-assisted exponential enzymatic amplification(T-EXPEA) method for ultrasensitive detection of miRNA based on threeway junction(3-WJ) structure driven,provided a rapid, one-step and high-efficient isothermal amplification method. Second, the secondary MB utilized exponential enzymatic amplification(SUMB-EEA) method has been used for one-step sensitive detection for miRNA.In the first approach, a novel T-EXPEA method for ultrasensitive detection of miRNA based on 3-WJ structure driven has been reported. In this assay, target miRNA can unfold hairpin probe and start the reaction, thus specifically form stable 3-WJ structure by hybridization with helper. Then it will produce triggers under the collective effect of polymerase and restriction endonucleases. Produced triggers initiate exponential enzymatic amplification(T-EXPEA) by bonding and unfold MB, which a series of new triggers will be produced to initiate T-EXPEA process once more and afford significant enhancement of fluorescence signals. The feature of our strategy lies in the T-EXPEA combining with 3-WJ structure has been utilized for fluorescence miRNA detection. It is worth noting that the sequence of the triggers in T-EXPEA part is the same to that of triggers generated from the3-WJ part. In addition, the design of restriction enzyme cutting sites using the same restriction enzyme(Nt.BbvCI) in hairpin probe and MB respectively, improved reaction efficiency cost-efficiently. This method can quantitatively detect sequence-specific miRNA in a dynamicrange from 10 aM to 10 pM with a detection limit as low as 7.8 aM.In the second approach, the SUMB-EEA method combined with 3-WJ structure has been used for one-step sensitive detection for miRNA. In this scheme, primers produced by 3-WJ structure initiate SUMB-EEA by bonding hairpin probe and generating a series of multiple new triggers to unfold secondary MB Utilized and afford significant enhancement of fluorescence signals. The feature of our strategy lies in MB has been secondary utilized for a significant fluorescence signal enhancement for fluorescence miRNA detection. SUMB-EEA method offers improved sensitivity and ultrahigh efficiency. This method can quantitatively detect sequence-specific miRNA-21 in a dynamic range from 10 aM to 1 pM with a detection limit as low as 3.5 aM.