| Small molecule compounds contains a wide variety of types,like the amino acid,sugar and nucleotide that exist in organism,all kinds of toxin,made by fungus,vitamins,and many synthetic compounds which are widely used in modern industry.For people's daily life and environment many of these compounds are toxins and pollutants,and they are filling almost all aspects of human life,which has obviously become a global public health problem,so it's of great value to realize a rapid and convenient method for small molecule compounds.Now,most detection of small molecule compounds are realized by classic chromatography methods like HPLC and GC,which have high sensitivity and specificity,but these methods always require assistance of sophisticated equipment and well-trained operator,with no potential to use them as point-of-care testing,well biosensor has broken some limitations of classic analysis methods,and has possessed detection characteristic of real-time signal output,high specificity,rapid and micro-sample requirement for different kinds of targets,which makes them easier to realize wide promotion and application.As a new small molecule compounds recognition bio-tool,transcription factor has gained attention from the field of analysis chemistry.By using its feature which could be away from target DNA after the recognition of its target molecules,people designed new type biosensors,and use protein modification to give transcription factor the ability to recognize other small molecule compounds,which expand the target range and is beneficial to further application in biosensing.Based on the recognition function of transcription factor and T7 RNA polymerase and Cas12 a signal amplification system,we designed two small molecule compounds biosensing platforms to realize a rapid,universal detection method.For that,we did the following work:1?Expression and purification based on prokaryotic system and property verification of TetR and Cas12 a.TetR can bind to ds DNA which contains a specific sequence,and form dimer on the sequence.By using gel electrophoresis,we identify the binding property of TetR which is obtained in our lab.Then,we identify the response property for its target molecule tetracycline,and also observe the response results at the existence of tetracycline,which shows a concentration-dependent reaction.At last,we find that different length of ds DNA will not affect the binding of TetR and its sequence,which matters to the design of biosensor.Based on the exploration of our lab,we also identify the binding of Cas12 a and sg RNA,specific and non-specific cleavage activity for ds DNA and ss DNA,to make sure that it can be used for the next experiments.2?Construction of small molecule compounds biosensing platform based on transcription factor and T7 RNA polymerase.The initiation of transcription of T7 RNA polymerase requires a recognition of promoter at first,then it goes downstream.By using the steric bulk of TetR that binds on the sequence,we design a switch for tetracycline,and using fluorescence signal generated by the combination of HBV and RNA aptamer Pepper which is produced by transcription as output,we construct a small molecule compounds biosensing platform,and work out the problem among recognition process,then realize a step-by-step detection method,and make characterization of the basic properties,and also realize visualization signal output of detection result.3?Construction of small molecule compounds biosensing platform based on transcription factor and Cas12 a.Facing the defect of the previous chapter's work,we make some promotion with the help of Cas12 a as the signal amplify module.Also,by using steric bulk of TetR that binds on the sequence,we design three different kinds of Cas12 a targets,which has different location and amount of TetR binding sites.The binding of TetR hinder the recognition of PAM by Cas12 a,we choose the one that is best hindered by TetR,and design a switch for tetracycline,which unleashes the Cas12 a target sequence at the existence of tetracycline,the unleashed target can activate the non-specific cleavage activity of Cas12 a,leads to the degradation of Reporter sequence and then unleashes free FAM group,the detection result will be outputted in form of fluorescence.After that,we optimize the concentration of protein and target sequence in reaction,to get a best detection effect.We also make characterization of basic properties of this platform,and we find that it has shorter reaction time,easier operation and better linearity calibration curve compared to platform based on T7 RNA polymerase,and can also realize visualization signal output of detection result. |
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