Novel Small-molecule Biosensing Methods Based On Regulation Of Transcription Factors And Cascade Signal Amplification

In recent years,environmental problems caused by small molecule pollutants have become increasingly prominent,so it is urgent to develop new environmental analysis and monitoring methods to meet the needs of environmental pollution control.Small molecule detection methods based on biosensing have the advantages of rapidity,simplicity,high sensitivity,and strong specificity,and play an important role in the detection of environmental pollutants.However,in order to achieve higher detection sensitivity and meet the needs of a wider range of small molecule detection,it is still necessary to combine more powerful signal recognition elements and signal amplification strategies to develop efficient small molecule biosensing analysis methods.Transcription factors(TFs)can achieve transcriptional regulation functions by binding with small molecule ligands and causing conformational changes to change the binding affinity with operon sequences.The types of TFs are very rich in nature,so they have a wide range of small molecule recognition,and are widely used in biosensing as small molecule signal recognition elements.In the signal amplification strategy,rolling circle transcription(RCT)based on T7 RNA polymerase can circularly amplify and transcribe long RNA chains of thousands of bases around a special circular template,and has a powerful nucleic acid amplification function;CRISPR-Cas12 a system is an RNA-guided DNA endonuclease that activates specific cleavage activity and efficient non-specific cleavage activity under the activation of target DNA,and has been developed into a powerful signal amplification tool for high-sensitivity in molecular diagnostic.In this thesis,we exploited the properties of transcriptional regulation of TFs,combined the signal amplification of RCT and CRISPR-Cas12 a system,to develop a novel method for the analysis of small molecule biosensing based on transcription factor regulation and signal amplification.Mainly include the following:(1)Small molecule detection method based on transcription factor TetR and rolling circle transcription signal amplification.The method utilizes the specific recognition of tetracycline molecules by the transcription factor TetR and the properties of allosteric regulation to regulate the RCT process.And the nucleic acid amplification function of RCT is used to amplify the response signal of the tetracycline molecule,so as to achieve the purpose of high sensitivity and specificity detection of the tetracycline.We designed a T7p-tetO-Pepper circular template,and explored the binding efficiency of TetR to the circular template and the inhibition efficiency of TetR on RCT.It is verified that this method can respond to tetracycline with high sensitivity and specificity,release the inhibition of RCT and amplify a large number of fluorescent RNA aptamer Pepper sequences with excellent fluorescence properties.Finally,the amplified fluorescent signal is used as the signal output to conduct qualitative and quantitative detection of tetracycline.The detection limit for tetracycline molecule is 46 nM,and the detection linear range is about 0.08-0.6 μM.(2)Small molecule detection method based on the transcription factor TetR and cascaded signal amplification.The method utilizes the specific recognition of tetracycline molecules by TetR and the regulatory function of TetR on the RCT process,and combines the nucleic acid amplification function of RCT and the signal amplification function of CRISPR-Cas12 a system to achieve cascade signal amplification in response to tetracycline molecules,so as to achieve higher detection sensitivity of tetracycline molecules.We designed a T7p-tetO-crRNA circular template,and explored the binding efficiency of TetR to the circular template,and compared the inhibition efficiency of TetR on fluorescence signal in one-step and two-step methods.Finally,we verified that this method can respond to tetracycline with higher sensitivity,and release the inhibition of RCT under the action of tetracycline molecule to generate a large number of crRNA sequences to achieve single signal amplification.After that,the trans-cleavage activity of CRISPR-Cas12 a system was further activated to achieve the double signal amplification.Finally,the fluorescence signal after double amplification is used as the output,and the detection limit of tetracycline molecule reaches 4.7 nM,and the detection linear range is about0.02-0.6 μM.This thesis provides a new idea for the biosensing analysis method of small molecules.