Research On Label-free Biosensing Of CRISPR/Cas12a

The outbreak of COVID-19 has raised major public health concerns.Developing a rapid and reliable diagnosis is critical to tracking and interrupting the spread of SARS coronavirus;Polychlorinated biphenyls（PCBs）are a common persistent environmental pollutant consisting of 209 congeners.Due to the highly toxic effects of PCBS,they can lead to thyroid dysfunction,endocrine disorders,cancer and other diseases.While,PCBS are not easy to degrade in the environment.Therefore,monitoring PCBS in the environment is of great significance.mi RNAs are a class of non-coding and endogenous short RNAs,which can regulate gene expression by participating in post-transcriptional processes.They are involved in many biological processes such as cell differentiation,apoptosis,proliferation and signal transduction.A large amount of clinical evidence shows that abnormal mi RNA expression is closely related to the occurrence and development of diseases,especially cancer.Therefore,its urgent to develop a simple method for rapid and sensitive nucleic acid detection.In this paper,CRISPR/Cas12a is combined with electrochemical,Raman and fluorescence assay for lable-free detection platforms.The main research contents are as follows:（1）Personal glucose meter（PGM）as a portable electrochemical device was utilized for sensitive detection of non-glucose targets:N-gene and PCB77,respectively.DNA hydrogel,which can respond to CRISPR/Cas system,was prepared for label-free encapsulating invertase.In the presence of targets,the repeated sequence for the activation of Cas12a was obtained due to the performance of RCA.Activated Cas12a can efficiently cleave multiple single-stranded linker DNAs on DNA hydrogels,thus releasing many invertase that can be used for PGM detection.With the amplification of RCA and CRISPR/Cas system,high detection sensitivity can be obtained even using portable PGM.The developed biosensor can be used for online monitoring.（2）Stimuli-responsive magnetic mesoporous silica microspheres Fe₃O₄@m Si O₂have been widely used in biosensor.In this work,we developed a CRISPR/Cas 12a system responsive Fe₃O₄@m Si O₂for label-free detection of cancer marker mi RNA21.A lot of methylene blue（MB）,as SERS signal molecule,was loaded into the mesopores of Fe₃O₄@m Si O₂.G-quadruplex/hemin complex,as a gate,was capped on the surface of Fe₃O₄@m Si O_2.Target mi RNA21 can initiate primer exchange reaction（PER）,whose product can activate CRISPR/Cas12a system.Thus,the activated Cas12a non-specifically cleaved the single-stranded DNA linked between G-quadruplex and Fe₃O₄@m Si O₂,resulting in the removal of the cap and the release of the MB.The supernatant was dropped on the surface of Zn O NRs@Au NPs,and MB signal was detected through SERS to realize the analysis of target mi RNA 21.Meanwhile,the G4/hemin complex in the supernatant can be used to catalyze the oxidation of ABTS for colorimetric detection.In this way,a label-free SERS-colorimetry dual mode biosensor was developed for highly sensitive detection of oligonucleotides.