Construction Of MiRNA Electrochemiluminescence Sensor Based On Co-Reaction Promoter Enhancement Strategy

Micro RNA（mi RNA）,as an endogenous non coding RNA,is directly related to the expression of many disease genes.Therefore,as a new biomarker,its rapid and ultra-sensitive detection of pith and moment for the pre-clinical diagnosis,prognostic and therapeutic of the disease.Electrochemiluminescence measurement technology has won extensive attention in scientific research circles because of its advantages of fast response,high sensitivity and low signal-to-noise ratio.This paper is committed to combining electrochemiluminescence analysis with biosensor to construct electrochemiluminescence biosensor and realize the rapid detection of mi RNA.In order to resolve the difficulty of low ECL signal intensity,different types of co-reaction promoters are introduced to construct ternary ECL system and heighten the preciseness of detection.So as to overcome the defectiveness of disorderly interference factors and acute background noise,a variety of nucleic acid amplification methods and CRISPR/Cas system are placed to complete the cyclic amplification of targets and the rapid switching of signals.The sensing platform is implemented on the interface of microfluidic paper chip,which greatly improves the simplicity of operation and reduces the experimental cost.In this paper,different co-reaction promoters were prepared,different nucleic acid amplification strategies were introduced,and multifaceted biosensors were established for ultra-sensitive detection of mi RNA.The major work was as follows:（1）A ternary electrochemiluminescence biosensor platform based on DNA Walker was manufactured for the detection of mi RNA-141.3D-r GO with excellent conductivity was prepared as the substrate,Au Pd NPs as the co-reaction promoter,and N-（4-aminobutyl）-N-ethyl isoluminol（ABEI）/H₂O₂ system was introduced.The circulating process was constructed by one-step DNA Walker nucleic acid amplification strategy to realize the in-situ accumulation of target concentration and improve the sensitivity of trace detection.The electrochemiluminescence characterization determined that the biosensor possessed good linear range and low detection limit,and can realize the ultra-sensitive monitor of mi RNA-141.（2）A CRISPR/Cas12a mediated paper based biosensor platform accompanied by 3D DNA Walker was organized for ultra-sensitive detection of mi RNA-141.The system was equipped with“On-Super on-Off”signal conversion characteristics.The redox reaction of g-C₃N₄NSs/K₂S₂O₈ system was accelerated by using Au Pd NPs as co-reaction promoter.As a biological means of signal amplification,3D DNA Walker realized the accumulation of intermediate DNA and the recycling of targets.Finally,CRISPR/Cas12a realized the detection of the target with its non-specific lateral branch cutting ability and response signal conversion.The designed biosensor hold satisfactory ECL linear response to mi RNA-141.（3）CRISPR/Cas12a paper-based biosensor platform triggered by hybrid chain reaction was constructed to detect mi RNA-141.Synthesized CO₃O₄@Au NPs as a co-reaction promoter,NPs polyhedron participates in the reaction of Ru（bpy）₃²⁺/TPr A system and enhanced ECL signal.The hybridization chain reaction process triggered by primers was designed for self amplification,and spherical nucleic acid（SNA）was further used to graft the phosphor onto the electrode interface to further amplify the signal.Finally,the signal annihilation was realized by CRISPR/Cas12a signal switch.The biosensor has excellent performance and can detect the target accurately and quickly.