| Accompanying the development of society,science and technology,biological analysis has penetrated in biology,chemistry,medicine,food,industrial and other fields.For decades,more sensitive,accurate,convenient and economic biosensing techniques have been the focus of biology and chemistry.Signal amplification-based fluorescence detection techniques,using various enzymes and nanoparticles,have been developed and became a research hot spot in the field of biological analysis.At present,there are some shortcomings in signal amplification fluorescence biosensors,such as multiple markers,complicated and so on.Developing novel signal amplification fluorescence detection techniques for solving more and more analytical problems and social crisis are still significant topics for researchers in biomedical and analysis chemical areas.In this thesis,a series of fluorescence signal amplifying detection platforms based on rolling circle amplification,enzyme-assisted cycling amplification and nanoparticles have been developed for quantitative analysis of nucleic acid and enzyme.The six chapters included in this dissertation are presented as following:The first chapter is the introduction which briefly introduces the principle and character of biosensing.It focuses on the classification and sensing mechanisms of fluorescence biosensor and reveals the recent developments of signal amplification-based fluorescence biosensor.Finally,we summarized the purpose,significance and the innovation of this thesis.The second chapter is linear DNA probe,as alternative to molecular beacon,for improving sensitivity of fluorescence biosensing platform for DNA detection using target-primed rolling circle amplification.Herein,we report a simple and homogenous fluorescence method for ultrasensitive DNA detection.It is based on rolling circle amplification(RCA)and fluorescence resonance energy transfer(FRET).Two short linear DNA probes were designed,which were labeled at the end with fluorophore and quencher,respectively.As an alternative to molecular beacon(MB),linear DNA probe was used in this RCA-based fluorescence strategy for DNA detection.The performance of linear DNA probes was compared with that of MB probe in this strategy.The results showed that linear DNA probe could effectively avoid the fluorescence quenching between neighboring signal probes,which would significantly improve the sensitivity of RCA-based fluorescence strategy.This method exhibited a high sensitivity toward target DNA with a detection limit of 0.7 aM,which was about 100-fold lower than that of the fluorescence strategy with MB as signal probe.This method provides a simple and convenient "mix-and-detect" protocol for homogeneous assay for sensitive detection of DNA and holds a great potential for early diagnosis in gene-related diseases.The third chapter reveals photoinduced electron transfer-based fluorescence quenching method for miRNA detection using target-primed rolling circle amplification.In the preceding chapter,it was found that only when the fluorophore-labeled probe and quencher-labeled probe self-assemble in head-to-head fashion on the RCA product,can the efficient FRET happen.Otherwise,there is no efficient FRET between fluorophore and quencher,and this would decrease the detection sensitivity.To address this problem,we designed the padlock DNA probe with "-CCC-" sequence.After RCA reaction,it could generate a long product with "-GGG-" sequence,which could be hybridized with thousands of fluorophore-labeled linear probe,thereby leading to efficient photoinduced electron transfer(PET)between fluorophore and "-GGG-".So the method can be used for detection of miRNA with high sensitivity,and the detection limit was estimated to be 6 aM(3σ).The fourth chapter demonstrates a one-step and dual-amplification strategy for highly sensitive fluorescence detection of DNA,which is based on ExonucleaseⅢ-assisted target recycling amplification coupled with liposome-assisted amplification.In this proposed strategy,a dumbbell-shaped DNA probe is designed to integrate target binding,magnetic separation and signal response.In the presence of target DNA,the multifunctional dumbbell probe can initiate exonuclease Ⅲ-aided target recycling amplification,and meantime generate a large number of fluorescein encapsulated liposomes.The developed method offers very high sensitivity due to primary amplification via numerous FAM from a liposome and secondary amplification via target recycling amplification.The detection limit of the proposed method can reach 4 aM,which is much lower than that of the Exo Ⅲ-aided target recycling technique applied for DNA quantification without liposomes amplification.Moreover,the dual-signal amplification process can be completed in one step.So,this method provides a simple and low-cost approach for sensitive detection of DNA and holds a great potential for early diagnosis in gene-related diseases.The fifth chapter discusses a facile and sensitive fluorescence assay of terminal deoxynucleotidyl transference activity and inhibition via in-situ formation of copper nanoclusters using enzymatically-generated DNA as template.In the presence of the primer and substrate(dATP and dTTP),TdT catalyzes the repetitive addition of dATP or dTTP to elongate the 3’-OH end of DNA primer and consequently produce long AT-rich dsDNA sequences.The dsDNA can serve as an excellent template for fluorescent CuNCs synthesis.Thus,TdT activity can be facilely detected with a simple fluorescence reader.This method has a linear detection range from 0.7 U/μL to 14 U/μL with a detection limit of 0.06 U/μL.The sensing protocol was applied to assay TdT activity in acute lymphatic leukemias cells.The protocol relies on the target-triggered formation of dsDNA polymers and in-situ formation of CuNCs with strong fluorescence intensity.This label-free approach is selective,simple,convenient and cost-efficient without any complex DNA sequence design or fluorescence dye label.The method not only provides a platform formonitoring activity and inhibition of TdT but also shows great potential in biological process researches,drug discovery,and clinic diagnostics.The sixth chapter reports a simple fluorescence strategy for sensitive detection of endonuclease EcoRI activity and inhibition using dsDNA-functionalized magnetic microspheres as probes.A series of dsDNA of different sequence compositions and a T-rich ssDNA were investigated for the formation of CuNCs.The fluorescence intensity was measured to determine the effect of each dsDNA and ssDNA.As a result,dsDNA,which was arranged alternately by adenine and thymine(AT-dsDNA),is found as the specific sequence which can act as a highly-effcient template for the formation of the fluorescent CuNCs.So we designed a dsDNA which contains the cleavage site corresponding to EcoRI endonuclease and AT-dsDNA,and the designed dsDNA was loaded on magnetic microspheres to form a dsDNA-functionalized magnetic microsphere probe.Upon the addition of the target endonuclease,the dsDNA molecules on the surface of the magnetic microsphere were cleaved.The AT-dsDNA cleaved products were released into the solution,which can be applied as an efficient template for fluorescent CuNCs.The endonuclease activity could be quantified by monitoring the fluorescence intensity with the detection limit 3×10-4 U/μL.This strategy is simple,rapid and "green"(environment-friendly)for sensitive detection of endonuclease EcoRI activity. |
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