Fluorescence Analysis For The Detection Of Microrna Based On Hairpin Nucleic Acid And Its Application In Cancer

MicroRNA（MiRNA）plays an important role in many biological processes,and is considered as a promising cancer biomarker.MiRNA is a class of endogenous noncoding single stranded RNA,which exists in plant and eukaryotic cells of animal.MiRNA is involved in the regulation of more than 60%of the protein coding genes in human,and plays a key role in many physiological processes.There is growing evidence that miRNA has become a significant biomarker in early clinical diagnosis,abnormal expression of miRNA in tissues and blood is closely related to many kinds of human diseases,such as cancer and cardiovascular diseases.With the further research on the mechanism of miRNA,this kind of small molecule will become an important breakthrough for us to better understand and conquer human diseases.Our main research is as follows:1.We develop a rapid and sensitive miRNA assay on the basis of exponential isothermal amplification in combination with the hairpin probes.The binding of target miRNA with a single stranded DNA template initiates exponential isothermal amplification reaction（EXPAR）and generates the universal triggers which are complementary to the 3′protruding end of hairpin probe1.These triggers function not only as the primers to unfold the hairpin probes through catalysed hairpin assembly,generating distinct fluorescence signals,but also as the primer to initiate the next EXPAR.This assay exhibits high sensitivity with a detection limit of 0.1 zmol through the detection of amplification product.According to the primer,and two probes labeled with different fluorescent dyes were designed according to miR-21.This hairpin probe-based circular exponential amplification assay holds a great promise for further application in biomedical research and early clinical diagnosis.2.In this work,a microfluidic paper-based fluorescent sensor device was constructed for quantitative detection of miRNA and determination of the expression of folate receptor（FR）on the cell surface.It is worth pointing out that simultaneous detection of two biomarkers in cancer was achieved through dynamic self-assembly of T-shaped duplex structure.T-shaped duplex structure can respond to specific triggers and convert to an output due to the conformation changes,it is meaningful to fabricate T-shaped duplex structure into powerful biosensors that can monitor chemical stimuli.Moreover,lamellar MnO₂ nanosheets were synthesized,which exhibited excellent quenching ability via adsorbed fluorophore-labeled single stranded DNA.This newly designed biosensor provided a sensitive detection of miR-21 from 0.01 to 5.0 fM with a relatively low detection limit of 0.0033 fM.As biological markers for disease diagnosis,the detection of FR and miRNA will provide a new approach and therapeutic tool for early clinical diagnosis.3.In combination with the characteristics of exonuclease III（Exo III）,a facile and highly sensitive isothermal cycle amplification based on Exo III for the detection of miRNA was developed by means of fluorescence analysis.In addition,the applicability of this method in complex samples was demonstrated by the analysis of RNA extracts from cancer cells.Therefore,this method provides a strong inspiration for biological research and effective diagnostic experiments.