Dnazyme-based Signal Amplification For Colorimetric/Fluorescence Assay Of Multiple MiRNA

miRNA is a short,non-coding,endogenous RNA molecule that is a key endogenous regulator of gene expression and reverse transcription.It serves as an indicator of physiological processes such as cell differentiation,proliferation and apoptosis.It is inseparable from the process of tumor growth and inhibition.Therefore,the detection of miRNA is of great value for disease prevention,clinical diagnosis and cancer treatment.Due to its small size,low abundance and high sequence homology among family members.The development of suitable methods for detecting miRNA is highly challenging.Traditional miRNA detection methods include Northern blotting,real-time polymerase chain reaction?RT-PCR?and microarrays.Although these methods can partially meet the requirements of detection,they are low in sensitivity,expensive in equipment,time consuming and complicated in operation.These situations limit their widespread use in biomedical applications.DNAzyme is a single-stranded nucleic acid that can catalyze many chemical reactions.It possesses unique property,such as high stability,low non-specific adsorption,good catalytic activity and easy preparation.It is widely used as a signal amplifier for enzyme-free catalytic reaction in the high sensitivity detection of DNA,protein and metal ions.In this paper,we develop two fluorescence/colorimetric sensing methods for simultaneous analysis and detection of target multi-miRNA,based on the catalytic amplification of DNAzyme.1.DNAzyme-based Signal Amplification for Colorimetric Assay of Multiple miRNAThis chapter constructs a dual DNAzyme catalytically amplified colorimetric sensing platform.It is used the combination of 8-17 DNAzyme and G-tetramer DNAzyme to form a substrate chain.The two split enzyme chain ends are part of complementary to the substrate chain.Due to the mismatch of conformation,the cleavage reaction cannot be performed.When the target miR-122 is present,the conformation of the DNAzyme is rearranged and causes autocatalysis to cleave the substrate chain.The active DNAzyme will automatically move to the adjacent substrate chain and begin the first step of cyclic amplification process.The released G-quadruplex and hemin form a G-quadruplex/hemin complex,it effectively catalyzes H₂O₂ to oxidize ABTS^2-to form a visually identifiable colored product.It is realized to detect miRNA through the second step of signal amplification In addition,multiple miRNA assays can be performed by altering the bases at the ends of the arms.This colorimetric method has the advantages of high specificity,simple operation and low cost.It has good practical availability.2.DNAzyme-based Signal Amplification for Suspension Microsphere Arrays of Multiple miRNAIn this chapter,the substrate chain of 8-17 DNAzyme labeled with fluorophore FAM and quenching group Dabcyl is first immobilized on four different sizes of polystyrene?PS?microspheres.The split DNAzyme was incubated with the corresponding PS microspheres to construct a suspension microsphere array based on DNAzyme catalytic amplification.Four miRNA chains miR-21,miR-122,miR-335and miR-155 were selected as detection targets.In the presence of the corresponding target miRNA,the activation of DNAzyme results in cleavage of the substrate chain on the corresponding PS microspheres,resulting in quenching of the group away from the fluorophore and fluorescence recovery of the PS microspheres.Release of the miRNA can further activate the DNAzyme to achieve automatic continuous cleavage of the substrate chain.It achieves signal amplification of the enzymatic fluorescent signal.Next,the fluorescence signals of different sizes of PS microspheres is recorded by flow cytometry,and achieves simultaneous identification and detection of multiple miRNA.