Research On New Methods Of Sensitive Detection Based On Nucleic Acid Circulation Circuit And CRISPR/Cas12a

Constructing simple and sensitive new detection methods has always been a hot spot in the field of analysis,detection and sensing.The signal amplification strategy can improve the detection sensitivity,but the amplification process usually involves a variety of tool enzymes,resulting in increased costs,complicated operation,and false positive signals.The cascade nucleic acid circuits that do not rely on the function of the protease,and show potential applications in the development of high-sensitivity biosensing technology methods based on signal amplification reaction autonomously through the base complementary pairing rule.Beyond its extraordinary genome editing ability,the CRISPR/Cas（clustered regularly interspaced palindromic repeats/CRISPR-associated proteins）systems have opened a new era of biosensing applications due to its high base resolution and simple and fast the detection process.Therefore,we construct fluorescent biosensing platforms to realize the sensitive,accurate and rapid detection of biomolecules with the help of the nanomaterials based on the cascade nucleic acid circuit and the CRISPR/Cas system.The main researches are presented as following:1.Double-signal mode based on metal-organic framework coupled cascaded nucleic acid circuits for accurate and sensitive detection of serum circulating mi RNAs.This chapter mainly constructs a dual-signal mode based on a metal-organic framework coupled cascade nucleic acid circuit for accurate and sensitive detection of cmi RNA in serum.In this strategy,catalyzed hairpin assembly（CHA）and hybrid chain reaction（HCR）constitute enzyme-free cascade nucleic acid circuits,which achieving multiple amplification and improving detection sensitivity.A magnetic bead（MB）with recognition and capture probes was used to enrich and separate the target cmi RNAs in serum,eliminating the influence of the complex serum environmental media.Benefiting from the cascaded nucleic acid circuits,magnetic enrichment and separation,and dual-signal mode,a high signal to noise ratio was achieved,and sensitive mi RNA detection down to 0.8 a M was realized.In addition,compared with q RT-PCR and NGS assay,the proposed method avoids PCR complex processing steps and rigorous experimental conditions,and economizes time and cost.The results showed that the method provides a sensitive and accurate approache for the quantitative detection of circulating mi RNAs in real samples by a non-invasive analysis mode.2.Simple and rapid detection of DNA methyltransferase activity based on target regulation CRISPR/Cas12a sensor.The chapter mainly constructed a simple,rapid and sensitive method based on the target regulation of CRISPR/Cas12a sensor for the detection of DNA methyltransferase activity.In this method,the characteristics of CRISPR/Cas12a reverse cleavage and the specific cleavage of Dpn I improve the signal to noise ratio and sensitivity with a detection limit as low as 2.15×10^-4 U/m L.In addition,other tool enzymes were not involved,which avoids false positive signals,complicated operations and high costs caused by amplification reactions.The proposed sensor does not need to strictly control temperature changes,and there is a small interference in the background signal.These results indicate that our method provides a platform for monitoring Dam MTase activity,which show great potentials in biological process researches,drugs discovery and clinical diagnostics.