Isothermal Signal Amplification Technology Is Used In Nucleic Acid Detection And Its Application In Molecular Logic Gates

It has been found that nucleic acids are promising biomarkers,which have the potential to realize the early diagnosis and treatment of certain diseases.The emergence of isothermal amplification provides a new method for efficient,rapid and sensitive detection of nucleic acids.Combining the isothermal amplification of DNA strand displacement reaction and tool enzymes with optical analysis methods,this thesis constructs a series of novel isothermol amplification strategies,which significantly improve the sensitivity for determination of nucleic acids.In addition,those isothermal amplification strategies provide new perspectives and methods for fabricating biological logic circuits and have great potential in the assembly of DNA nanostructures,construction of amplified biosensing platforms and molecular logic circuits.This thesis carried out the following four aspects of work: 1.Self-assembly of hyperbranched DNA structures for detection of mi RNA and logic operations.Based on catalytic hairpin assembly(CHA)and hybridization chain reaction(HCR),this work proposed strategy for self-assembly of hyperbranched DNA structures.The formed hyperbranched DNA structures can be used as an enzyme-free amplifier for sensitive detection of mi RNA-122 by chemiluminescence resonance energy transfer(CRET),achieving a detection limit of 0.72 pM.The proposed method also exhibits a good selectivity,which can well disdinguish single-base mismatched mi RNA sequence.In addition,this paradigm achieves a logic operation through constructing a cascade of DNA nanocircuitry with a feedback mechanism.2.Cross-catalytic hairpin assembly-based strategy for detection of DNA with exponential signal amplificationThis work developed a cross-catalytic hairpin assembly method(C-CHA)based on toehold-mediated strand displacement reaction.Integrating with CRET strategy,this method achieves exponential amplification of DNA.The background noise is effictively reduced due to the introduction of Dabcyl,which significantly improves the signal-to-noise ratio and sensitivity.The proposed method not only realizes the exponential amplification efficiency with a low detection limit of 0.67 pM,but also can be acted as multi-level circuits.3.Enzyme-free catalytic self-assembly of hyperbranched DNA structures for amplified analysis of miRNABased on toehold-mediated strand displacement reaction,a branched catalytic hairpin assembly(b-CHA)technology is presented.This system took Y-shaped hairpin trimers as precursors.In the presence of target DNA,the DNA strand displacement reaction was initiated,resulting in the self-assembly of hyperbranched DNA structures.Furthermore,this DNA catalytic device can be readily utilized to fabricate amplified biosensing platform via fluorescence resonance energy transfer(FRET),achieving sensitive detection of miRNA-155.And the proposed method exhibits good selectivity,which has been applied to analyze the nucleic acids in complex biological samples.4.Magnetic separation-based chemiluminescence imaging array for simultaneous detection of multiplex miRNAs via DNA strand displacementBased on DNA polymerase/NEase-assisted amplification technology,this work fabricates a novel chemiluminescence imaging array for simultaneous detection of multiplex miRNAs.The results show that this method has a wide linear detection range and high sensitivity.The detection limits for three miRNA are all at femtomolar level.Furthermore,this method can distinguish single-base mismatched miRNA,showing good specificity.And it also can be successfully applied to the analysis of serum samples.In addition,the magnetic beads used in the work can be recycled to reduce the analysis cost.This strategy has broad prospects in biosensing,disease diagnosis,and biomedicine.