Research On The Application Of Rolling Circle Amplification Technology In The Detection Of Biomolecules

Nucleic acid amplification is a very important technology in biology and medicine.Rolling ring strategy is an efficient and attractive simple method for amplifying nucleic acids,biological macromolecules and signal amplification.This magnification strategy as a platform has developed rapidly.Rolling ring strategy is an efficient and attractive simple method for amplifying nucleic acids,biological macromolecules and signal amplification.This magnification strategy as a platform has developed rapidly in many ways.Circulating tumor cells?CTCs?are cells that enter the circulatory system through the tissue matrix body and form metastases in important distal organs.In addition to the genome and proteome,tumor cells are information-carrying freighters of interest regarding tumor growth.Tumor cells are the source of malignant tumor metastasis.Therefore,cell sensing of CTCs has become an important issue in understanding its role in disease development and providing diagnostic tools to guide treatment.Bioactive substances play an important role in cell proliferation and differentiation,tissue metabolism and heredity of living body.Therefore,the detection of bioactive substances is of great significance in the field of clinical diagnosis and targeted therapy.The establishment of a simple,rapid and sensitive detection method has attracted more and more attention,so it has become the basic and hot research object in the field of biochemistry.We propose a simple method to synthesize RNA membrane nanostructures with controlled capture and cytosensing by aptamer and luminol-AuNPs?luAuNPs?.The modifications of-NH₂ group can make the RNA membrane chemically stable in the serum.The prepared RNA membrane nanostructures show interesting capture efficiencies toward Ramos cell due to the effect of aptamer-ligand interactions on the nanostructures.Additionally,a novel enhance method is employed by3-aminopropyl-triethoxysilane?APS?for luAuNPs electrochemiluminescence?ECL?,which has never been reported.A hyper-dendritic rolling circle transcription?HRCT?is presented herein,which consists of only one circular nucleic acid template that contains an identification sequence of the analyte RNA,a sequence identical to the molecular beacon modified with a fluorophore/quencher pair,and a sequence identical to the loop region of the coadded hairpin structure,can stably coexist until the introduction of an analyte RNA to trigger a cascade of RCT events,leading to the self-sustained assembly of hyper-dendritic RNA structures.We employ stem-loop-structured fluorescent molecular beacon to transduce the autonomous operation of this RCT machine.The system can achieve ultrasensitive detection of target RNA.The electrochemiluminescence intensity is proportional to the number of target cells.The dynamic range is 50-700 cells,and the detection limit of target cells can be as low as 50 cells.The platform of RNA membrane,based on rolling circle transcription,use oligonucleotides for cell-specific capture and release and delivery of the anticancer drug doxorubicin?DOX?.Firstly,using the principle of DNA self-assembly technology,the RNA membrane was formed by rolling circle transcription method.Introducing the Ramos aptamer,the prepared RNA membrane nanostructures show interesting capture efficiencies toward Ramos cell due to the effect of aptamer-ligand interactions on the nanostructures.Additionally,based on the detection of luAuNPs electrochemiluminescence?ECL?system,a novel enhancing method is employed by3-aminopropyl-triethoxysilane?APS?for luAuNPs ECL,which has never been reported,so as to improve the sensitivity.The ECL intensity was proportional to target cells amount,and the dynamic range was 50-5000 cells.A hyper-dendritic rolling circle transcription?HRCT?is presented herein,which consists of only one circular nucleic acid template.It can stably coexist until the introduction of an analyte RNA to trigger a cascade of RCT events,leading to the self-sustained assembly of hyper-dendritic RNA structures.We employ stem-loop-structured fluorescent molecular beacon to transduce the autonomous operation of this RCT machine.The system can readily achieve ultrasensitive detection of target RNA.With this simple method,miRNA-21 can be quantitatively detected down to 1×10^-1818 M.By the design of reduced thiol-cleavable disulfide bond in linker DNA,which can be cleaved by the exchange reaction of thiol ligands,the linker section in DNA is released and the HRCT principle is successfully applied to display excellent selectivity and sensitivity for reduced thiol.