Design And Bioanalytical Applications Of Nucleic Acid Fluorescent Probes

Nucleic acid fluorescent probes possess the advantages of high sensitivity,good stability,high specificity,and simple design,thus have been widely used for the the detection of a wide ranges of targets,such as DNA.metal ions,small molecules,proteins,and even whole cells.However,the practical application of nucleic acid probes still has some limitations,such as high cost,complex operation,unsatisfied reproducibility,and limited functions.To address these issues,this thesis designs two label-free nucleic acid fluorescent probes and a multifunctional nucleic ac:id fluorescent probe,and explore their applications in bioanalysis field.The detailed works are as follows:1.A facile label-free sensing method is developed for the one-step and highly sensitive fluorescent detection of DNA,which couples a trimethyl-substituted naphthyridine dye(ATMND)with the exonuclease Ⅲ(Exo Ⅲ)assisted cascade target recycling amplification strategy.In the absence of target DNA.the DNA hairpin probe with a C-C mismatch in the stem and more than 4 bases overhanged at the 3’ terminus could entrap and quench the fluorescence of ATMND and resist the digestion of Exo Ⅲ.thus showing a low fluorescence background.In the presence of the target,however,the hybridization event between the two protruding segments and the target triggers the digestion reaction of Exo III,recycles the initial target,and simultaneously releases both the secondary target analogue and the ATMND caged in the stem.The released initial and secondary targets take part in another cycle of digestion,thus leading to the release of a huge amount of free ATMND for signal transducing.Based on the fluorescence recovery,the as-proposed label-free fluorescent sensing strategy shows very good analytical performances towards DNA detection,such as a wide linear range from 10 pM to 1 μM,a low limit of detection of 6 pM,good selectivity,and a facile one-step operation at room temperature.Practical sample analysis in serum samples indicates the method has good precision and accuracy,which may thus have application potentials for point-of-care screening of DNA in complex clinical and environmental samples.2.A label-free ratiometric fluorescence aptasensor is developed for the rapid and sensitive detection of cocaine in complex biofluids.The fluorescent aptasensor is composed of a non-labeled GC-38 cocaine aptamer which serves as a basic sensing unit and two fluorophores,2-amino-5,6,7-trimethyl-1,8-naphthyridine(ATMND)and SYBR Green I(SGI)which serves as a signal reporter and a build-in reference,respectively.The detection principle is based on a specific cocaine mediated ATMND displacement reaction and the corresponding change in the fluorescence ratio of ATMND to SGI.Benefited from the high affinity of the non-labeled aptamer,the good precision originated from the ratiometric method,and the good fluorescence quantum yield of the fluorophore,the aptasensor shows good analytical performance towards cocaine detection.Under optimal conditions,the aptasensor shows a linear range of 0.10 to 10 μM and a low limit of detection of 56 nM,with a fast response of 20 seconds.The low limit of detection is comparable to most of the fluorescent aptasensors with signal amplification strategies and much lower than all of the un-amplified cocaine aptasensors.Practical sample analysis in a series of complex biofluids,including urine,saliva and serum,also indicates the good precision,stability,and high sensitivity of the aptasensor,which may have great potential for the point-of-care screening of cocaine in complex biofluids.3.A multifunctional nucleic acid fluorescent nanoprobe is synthesized and applied for the fluorescence recognition and imaging of cancer cells,as well as their chemo-photothermal therapy.The multifunctional probe is designed with a core@shell structure,in which the polydopamine nanospheres with strong photothermal property serves as the core,and aptamer cross-linked nanogel serves as the shell.The latter could recognize A549 cancer cell from other cells,transfer the loaded doxorubicin into the cancer cell,and release the chemicals into the cell under the irradiation of NIR light.Benefited from the synergistic interaction of chemo-photothermal therapy,more than 50%of the cancer cells can be killed,better than the treatment by only chemical or photothermal therapy.The smart multifunctional nucleic acid fluorescent probe has explored a new avenue for targeted imaging and synergetic treatment of cancer.