| Background : Testosterone is a kind of endogenous androgen in organisms.Due to excretion,accumulation and abuse of synthetic testosterone,testosterone can accumulate in food,water and soil.Excessive intake of exogenous testosterone can affect human reproductive health.Therefore,it is of great practical significance to develop a convenient and effective detection optimization method for the rapid detection of testosterone in the environment(such as water samples,food).Compared with antibodies,nucleic acid aptamers are better small molecule specific trappers with the advantages of rapid production,strong stability and small size.Fluorescent biosensor also has the advantages of simple operation,high sensitivity and high-throughput bioanalysis potential,so it has become one of the most popular design methods of aptamer-based sensing system.The detection method of unlabeled nucleic acid aptamer biosensor has the advantages of time-saving,labor-saving,universal,simple and less modification to the structure of aptamer and target.As an important recognition element of the sensor,nucleic acid aptamer can also improve the sensitivity by truncating and other ways to improve the availability of aptamer.Objectives: The aim of this project is to design an easy-to-use,easyto-use,and cost-effective fluorescent biosensor based on testosteronespecific nucleic acid aptamers for the determination of testosterone content in different solvent systems.At the same time,the truncated nucleic acid aptamer is designed.The binding affinity and conformational change between the truncated nucleic acid aptamer and testosterone are explored.This study lays a foundation for better aptamer testosterone detection applications.Methods:(1)This project predicted the secondary structure of the aptamer,and used the flexible docking mode of autodock to conduct molecular docking of the T5 aptamer and testosterone to explore the optimal conformation and intermolecular interaction.The conformational changes during the binding process were investigated by UV spectroscopy and circular dichroism spectroscopy(CD).(2)Combined with the fact that SYBR Green I fluorescent dye can be inserted into DNA and the testosterone nucleic acid aptamer has strong specificity for testosterone,a label-free fluorescent biosensor was constructed.At the same time,the linearity,accuracy,selectivity,recovery rate and sensitivity of the test method were verified.(3)This topic used isothermal calorimetry to verify the affinity of the truncated T5G2 aptamer with testosterone,and used ultraviolet spectroscopy,circular dichroism,molecular docking,etc.to explore the conformational changes during the binding process.Results:(1)Autodock molecular docking showed that the interaction between nucleic acid aptamer and testosterone was mainly through intermolecular energy,van der Waals force,hydrogen bond and dissolution force.The contribution of electrostatic force is small.Circular dichroism spectra show that the aptamer of T5 nucleic acid is a mixed conformation of B-type double-stranded conformation and antiparallel Gquadruplex.After the addition of testosterone,because B-DNA is very sensitive to show binding changes,CD did not observe a significant change in the CD spectrum of B-DNA after the addition of testosterone.There is no obvious shift in the intensity of negative band and positive band,and there may be no intercalation between nucleic acid aptamer and testosterone,but the action mode of groove binding is adopted,and the conclusion of circular dichroism spectrum is also verified by UV spectrum.It lays a theoretical foundation for the quantitative method in the next chapter.(2)based on SG fluorescent dye and 86-base sequence testosterone specific aptamer,an unlabeled nucleic acid aptamer fluorescence biosensor was constructed to detect testosterone in water samples conveniently,simply,accurately and effectively.The lowest detection limit of the fluorescence system for testosterone is 0.273 n M,the linear detection range is 0.91～2000n M,and the linear correlation coefficient is 0.9521.In the detection range,the linear fitting of this technique is good,and the selectivity,accuracy and recovery are good.(3)the original long base was truncated,and the 30-44 base sequence was selected as the new suitable ligand,and the interaction mode was verified by isothermal calorimetric titration,UV spectroscopy,circular dichroism spectroscopy,molecular docking and so on.After fitting,the Kd value was determined to be 1n M.Compared with the 86 mer aptamer,the affinity of T5G2 aptamer was improved.-T Δ S <0 indicates that it is a process of spontaneous binding,usually a conformational change.UV,circular dichroism and autodock docking also showed that the structure of T5G2 aptamer itself was a parallel quadruplex structure.After the addition of testosterone,there was an interaction between T5G2 aptamer and testosterone,the main form of interaction was groove binding,and the main interaction forces were hydrogen bonding force and hydrophobic force.However,its specificity and quantitative use still need to be further studied.Conclusion: the binding of T5-specific aptamer rich in 86 bases to testosterone was verified by circular dichroism spectrum,ultraviolet spectrum and autodock molecular docking.It was analyzed for the first time that the way between the aptamer and testosterone may be groove binding,and the binding force is mainly hydrogen bonding force and hydrophobic force.Secondly,an accurate and convenient label-free fluorescent biosensor was prepared by using the original long T5 nucleic acid adaptation system,and the nucleic acid aptamer was successfully applied to the quantitative detection of testosterone in tap water samples.Finally,the aptamer of T5 nucleic acid was truncated,and the binding mode was verified by UV spectrum,circular dichroism spectrum,isothermal calorimeter and molecular docking,and the Kd value was fitted.A 15-base aptamer with strong affinity was obtained,which reduces the cost and shortens the base sequence.Conclusion: This subject verified the binding of the 86-base-rich T5-specific aptamer and testosterone through circular dichroism spectroscopy,UV spectroscopy,and autodock molecular docking.The first analysis of the way between the aptamer and testosterone may be a groove binding,the binding force is mainly hydrogen bonding force and hydrophobic force.Secondly,an accurate and convenient label-free fluorescent biosensor was prepared by using the original long T5 nucleic acid aptamer,and the nucleic acid aptamer was successfully applied to quantitatively detect testosterone in tap water samples.Finally,the T5 nucleic acid aptamer was truncated in this project,and the binding method was verified by ultraviolet spectroscopy,circular dichroism spectroscopy,isothermal calorimetry,molecular docking,etc.,and the Kd value was determined,and the affinity was successfully obtained.The stronger nucleic acid aptamer containing 15 bases reduces the cost and shortens the base sequence. |
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