A Study Of A Fluorescent Sensing Model Based On The Self-assembly Of G-quadruplexes And Aptamer Hairpins

With the development of interdisciplinary research in computer science,life science,material science,electronic technology and nanotechnology,it has laid the foundation for the development of bioinformatics.As a branch of bioinformatics,biological computing has more powerful parallel and storage capacity than turing machine.And the biological molecules have excellent recognition ability,small physical scale and powerful operability,which also provide the possibility for biological computing.This logical relationship between biomolecules is not only widely used in molecular computers,but also one of the core research directions of biosensors.It is very important to develop simple,rapid,multiple and specific biosensors for water monitoring,soil detection,food safety and medical disease investigation.Among the new sensors reported so far,aptamer sensor has been widely used in the identification ofdrugs,proteins,small molecules and cells due to its high affinity,excellent selectivity,strong thermal stability and easy synthesis.Moreover,the G-rich sequences in many biological functional regions of the genome suggest that the G-quadruplexes have many funetions.The main work of this thesis is as follows:Firstly,we described the research background and significance of biological computing and biosensor,and classified the biosensors according to their functions,including the biosensors for DNA and RNA detection,specific protein,carbohydrate and lipid detection and constructing DNA logic gates.We also introduced the development of DNA computing and the research status and develop trend of the fluorescent labeling technology.Then the new nano materials and key detection technologies,including DNA aptamer,G-quadruplex,target cycle and signal amplification technology based on enzyme mediated and strand replacement were induced.These contents provided theoretical basis and technical support for our model design and experimental verification.Subsequently,we constructed a fluorescent aptamer sensing model based on G-quadruplex and hybridized chain reaction(HCR)for Ochratoxin A(OTA)detection.Firstly,the corresponding DNA hairpin structure was designed with the help of the simulation software NUPACK,and the Toehold length,experimental temperature and OTA concentration were optimized with the detection of the target product yield in solution.Secondly,we combined the thermodynamic model with dynamic programming algorithm to estimate the minimum free energy(MFE)of nucleic acid structures in different rings,so as to analyze the thermodynamic properties and stability of the designed chains.Thirdly,we used Visual DSD to design the whole simulation experiment process according to the syntax rules.Before the experiment,we verified the theoretical feasibility of the designed model.Based on that,we verified model by biological experiments.According to the orthogonal experiment,the initial conditions were obtained and the feasibility of the experiment was verified.What's more,the experimental conditions were optimized,including K+ concentration,hybridization reaction time,NMM concentration and reaction time.Then,under the optimal experimental conditions,the sensitivity,specificity and OTA detection range of the designed biosensor were explored.It is concluded that the biosensor can accurately identify OTA in other mycotoxins similar to OTA,and the proposed method also realized the accurate detection of OTA in actual samples(wine and wheat flour).Furthermore,we introduced the catalytic hairpin assembly(CHA)reaction amplification technology,and redesigned the fluorescence sensing model for OTA detection.We used the simulation software NUPACK to complete the sequence design and the optimization of the temperature and the corresponding chain concentration.The experimental process was simulated by visual DSD to verify the theoretical feasibility of the designed model.The initial Ochratoxin A concentration and other reaction conditions were also simulated to simplify the follow-up biological experiment steps.Finally,we summarized the two models designed for OTA detection,discussed the innovation and shortcomings in this study,and prospected the follow-up development of bioinformatics and biosensors.