Construction And Application Of Multi-functional Label-Free Nucleic Acid Aptamer Sensing System Based On Signal Amplification Strategy

This thesis focuses on the construction and application of label-free aptamer biosensors,which include nucleic acid aptamers as recognition elements,berberine as fluorescent probes.The signal amplification was achieved by assembling nanomaterials,nucleic acid tool enzymes,and hybrid chain reactions and multifunctional molecular logic gate technologies.The application for the detection of several environmental pollutants were also investigated.The main research contents are arranged as the following five chapters.1.overview of the characteristics of nucleic acid aptamers,the SELEX?systematic evolution of ligands by exponential enrichment?and its sensing technology,then focuses on the research progress of aptamer sensing system with the signal amplification in recent years.Finally,the original intention for the improvement,innovations,and the main contents of this thesis are presented.2.constructs the label-free nucleic acid aptamer fluorescence sensing system based on an enzyme-assisted amplification strategy with berberine as a fluorescent probe for the analysis and detection of tetrodotoxin?TTX?.TTX is able to specifically bind to the aptamer,that causes aptamer's configuration change from single-stranded to neck-loop structure.The fluorescence intensity of the sensing system changes because of the different interaction of berberine with the single-stranded oligonucleotide and neck-loop structure.The background signal of the detection system is fromthe single-stranded oligonucleotide bound berberine.Exonuclease I can specifically cut single-stranded DNA,and the addition of exonuclease I will significantly reduce the background signal for achieving the highly selective and sensitive detection of tetrodotoxin.Under the optimal experimental conditions,the linear range of tetrodotoxin is obtained from 0.03 n M to 6000 n M,and the detection limit is 11.0 p M,much lower than the previously reported methods.The control experiment was also conducted with enzyme-assisted and enzyme-free.The linear range of TTX in the enzyme-free system is from 0 n M to 500 n M with the detection limit as0.066 n M.The linear range and detection limit of enzyme-assisted system are much better than that of enzyme-free system.3.develops the label-free nucleic acid aptamer sensor with Mo S2 nanosheets.Mo S2 nanosheets were synthesized by hydrothermal top-down method using sodium molybdate and thioacetamide as the starting materials.This sensing system uses berberine as the signaling part for the detection of small molecules ATP.The specific binding of ATP to aptamer causes ATP-aptamer chain from a freely coiled state to a G-quadruplex structure.The fluorescence intensity of sensing system increases when ATP-aptamer changes from single-stranded oligonucleotides to G-quadruplexes.However,the intense background interference decreases the sensitivity for the detection of ATP.The aim of adding Mo S2 nanosheets to the above system,is to reduce the background interference,and then increase the detection sensitivity.Under the best experimental conditions,the berberine/APT-aptamer/MoS₂ detection system achieves the linear rangefrom 0.125 ?M to 38 ?M with the detection limit as 0.0402 ?M.The linear range with fluorescence anisotropy is from 0.0625 ?M to 45 ?M,with the detection limit as 0.0208 ?M.The spike recovery experiments show that ATP recoveries in serum samples was 94.8 %-106.8%?fluorescence method?and 93.2 %-108.5 %?fluorescence anisotropy?.4.constructs the label-free fluorescent nucleic acid aptamer sensing system using berberine as a fluorescent probe chain hybridization reaction and Mo S2 assisted signal amplification strategy for ultrasensitive detection of kanamycin.Adding kanamycin to the berberine/kanamycin-aptamer/Mo S2 system,the binding of kanamycin-aptamer changes the structure of aptamer into a double-stranded neck loop and triggers a chain hybridization cycle amplification reaction.A double-stranded structure with long-gap was formed,and the structural change resulted in a spatial separation from the surface of Mo S2 nanosheet.Therefore,the fluorescence intensity increased.In the absence of kanamycin,the ?-? stacking effect of single-stranded DNA and MoS₂ nanosheet decreased the spatial separation between aptamer and MoS₂ nanosheets,which quenched the fluorescence of the sensing system.The change of system fluorescence intensity before and after the addition of kanamycin is the origin of the sensing system.Under the optimal experimental conditions,the linear range of kanamycin is 0.1 p M⁹00 p M,and the detection limit is 0.06 p M.The spike recovery experiment shows that kanamycin spike recovery in the serum sample is 96.91%¹06.67%.5.is the further development of aptamer sensing system with a triple-output signal for the ultra-sensitive analysis and detection of Ag⁺and cysteine.The detection system is based on the formation of a stable C-Ag⁺-C i-motif structure when Ag⁺binds to Ag⁺nucleic acid aptamer.After cysteine?Cys?is introduced into the berberine/C-Ag⁺-C system,Cys and Ag⁺can form a stable Cys/Ag⁺complex and the C-Ag⁺-C double-stranded structure breaks apart,which leads to the fluorescence quenching of the sensing system.A simple,fast,and sensitive triple-output label-free fluorescent nucleic acid aptamer logic gate was constructed for the first time detection of Ag⁺and Cys.Under the optimal experimental conditions,the linear ranges of Ag⁺and Cys were 0 ?M 264.52 ?M and 0 ?M 70 ?M,and the detection limits as 0.079 ?M?3?/??and 0.030 ?M?3?/??.The spike recovery experiments showed that the recovery of Ag⁺in water and serum sample was 94.2%¹05.3%.