An Electrochemical Biosensor For Highly Sensitive Detection Of MicroRNA-377 Based On Strand Displacement Amplification Coupled With Three-way Junction

Nowadays,the complex biochemical properties of clinical samples,the low abundance of target substance make higher and higher requirements for inspection technology,and electrochemical sensors greatly adapted the development of clinical diagnosis and drug detection analysis due to the advantages of high sensitivity,fast response,low cost,simple operation and being used for microanalysis.In recent years,the researchers achieved to detect the target substances based on the electrochemical sensing platform and a variety of efficient amplification strategy.The structure,optical and electrical properties of nanostructure self-assembly provide a new opportunity for the new nanoelectrochemical sensing research.At the same time,with the development of molecular biology,nucleic acid amplification detection technology has developed rapidly.In this study,mi RNA-377 was detected on the electrochemical platform with strand displacement amplification and nano self-assembly.The main research contents were divided into two parts:1.Chain displacement reaction for the target substance.The template chain mainly comprises three regions: the target substance recognition region,the shear enzyme recognition region,and the downstream primer region.By such a design,the target material can be complementarily interacted with the template in the presence of a template owning a cleavage recognition site template chain for specific binding.In the presence of KF polymerase,d NTP under the conditions of continuous polymerization,resulting in double-stranded exposure to the site of cleavage and shear under the action of the shear enzyme,through continuous circulation,a large number of linkers are produced.The linkers can hybridize together with the capture probes and the three-way junction nanostructures respectively.2.Self-assembly of three-way junction nanostructure.The three strands which are complementary mutually are placed in the solution of the appropriate ion concentration and they are bound to form a stable three-way junction nanostructures by hybridization.The non-complementary part of one strand connect the product of SDA reaction product resulting from the strand displacement reaction forms the composition and then attaches the composition to the capture probe.The electrochemical signal is obtained by catalyzing the reaction of the substrate ?-naphthol by combining the biotin-modified Y-type nanostructures with streptavidin linked with alkaline phosphatase.The linear detection range of mi RNA-377 was between 1 f M and 1 n M,and the detection limit was 0.68 f M.Good specificity,good reproducibility,reproducibility and stability were also obtained through experimental comparison.The electrochemical sensors put strand displacement and Y-type nanostructures into amplification techniques for realizing high sensitivity and high specificity detection of target substances and successfully applied to the detection of actual samples for medical field.It also provides a new technical support for early diagnosis and the application of highly sensitive detection of mi RNAs.