Studies On New Fluorescent Analysis Methods And Application Based On Click Chemistry And Nucleic Acid Signal Amplification Technologies

Take advantages of high sensitivity,good selectivity,simple operation,low cost and in situ measurement,biosensing technique has been widely applied in the drug,food analysis,clinical diagnosis,biological medicine,environmental monitoring and so on.Nucleic acid signal amplification technology based on a polymerization amplification or digestion cycle restriction methods,which can significantly improve the sensitivity of analysis.Construction of the biosensing analysis methods using the nucleic acid amplification techniques has an important significance in the analysis performance.Click chemistry with mild reaction condition high yield,no by-products generated and does not require specific purification process,has a broad application prospects in drug discovery,polymer synthesis,DNA ligation,cell imaging and so on.Nanomaterials have special optical and electrical properties.They provide a new way to build a new biological sensing system combining nanomaterials and click chemistry to construct the optical bioanalysis.Based on nucleic acid signal amplification and DNA-templated click chemistry,this thesis has developed three new fluorescent biological sensing methods for quantitative detection of nucleic acid and copper ions.The main contents of this work can be concluded as follows:Part one:A simple and convenient fluorescence method based on DNA-templated click reaction and strand displacement signal amplification has been established for the rapid detection of Cu2+.The DNA-templated click chemistry was triggered by addition of the Cu2+ in the system,making a chemical ligation of the probe H1 and keeping it in a stable hairpin structure.As a result,the signal probe H2 binds with a fluorescent molecule ThT to generate strong fluorescence signal.On the contrary,the hairpin probe H1 can hybridize with signal probe H2 through endless chain substitution reaction in the absence of Cu2+.In result,the G4 structure of H2 was damaged and failed to combine with ThT,which greatly reduced the fluorescence background signal Under optimized conditions,the Cu2+ can be sensitively and specifically detected with a linear range of 5.0×10-7 mol/L?2.0×10-5 mol/L with a low limit of detection of 2.23×10-7 mol/L(S/N=3).In addition,the developed assay also serves as a detection platform for Cu2+in the river water with satisfactory results.Part two:A novel fluorescence polarization(FP)assay based on DNA-template click reaction and silica nanoparticles(SiO2)-assistant FP signal amplification was developed for highly sensitive and selective detection of Cu2+.In this work,Cu2+ Was reduced to Cu+ in the presence of sodium ascorbate firstly.Then,Cu+ catalysis the chemical ligation of alkyne and azide group which bearing the duplex DNA probe P1-P2 respectively.A new biotin-labeled DNA probe P3 would hybridize with P1 to induce DNA sequence displacement to form P1-P2-P3 complex.After that,this complex would combine with streptavidin-coated SiO2(SA-Si02)via the specific interactions of biotin and streptavidin generating a strong fluorescence polarization signal While a weak FP signal was obained without Cu2+,becauce P1 and P2 is unable to ligate.And the free FAM-P2 was obtained in solution after strand displacement reaction with P3.Under optimized conditions,the Cu2+ can be sensitively and specifically detected with a low limit of detection of 1.78 ×10-8 mol/L.Part three:A simple,homogenous label-free and ultrasensitive fluorescence method based on nucleic acid enzyme-assisted isothermal amplification and G-quadruplexes/ThT-triggered signal enhancement was established for detection of miRNAs.Unter an ingenious designs of hairpin probe and template probe,when the target is complementary hybridized with the hairpin probe,the enzymatic cascade signal amplification is initiated to produce a large amount of multicopies of target and G-quadruplexes.This resulted in a strong fluorescence signal when ThT embedded in the G-quadruplex to form G-quadruplex/ThT complex with the assist of K+.Taking the miRNA-261 as a model target,this approach was ultrasensitive according to the variation of fluorescence intensity and the detection limit is as low as 5.6×10-15 mol/L.At the same time,this method has high specificity and applicability in the complex environment.The result indicates that this approach should be a promising tool for biomedical research and clinical diagnostics.