The Research Of New Biosensing Mothed Based On DNA Self-Assembled Nanostructure

“Liquid biopsy”is an important tool for early noninvasive diagnosis of malignant tumor patients,and has become a research hotspot in the field of molecular diagnosis and bioanalytical chemistry.Circulating tumor DNA（ctDNA）,as one of the main targets of“liquid biopsy”,contains tremendous and stable biological information,and has important value in disease diagnosis,treatment and prognosis evaluation.However,the current detection motheds can not meet the needs of diagnosis under different environmental conditions,which limits the wide clinical applications.At the same time,the advances of nanotechnology and material science have led to rapid development of vitro diagnostic techniques in recent years,and many new nanomaterials have been successfully applied in the field of medicine and health,laying the foundation for further exploration.Moreover,the development of biosensing technology has also brought new impetus for the acquisition of biological information to meet different needs.Therefore,in view of some problems existing in the“liquid biopsy”,this study integrates DNA nano-assembly technology,material science and technology with surface plasmon resonance（SPR）and fluorescence resonance energy transfer（FRET）biosensing technology to develop a series of sensitive,specific and enzyme-free detection motheds of nucleic acids.The implementation of this study will help to expand the research ideas in molecular detection field,and provide more theoretical and practical evidences for enriching the methodology of“liquid biopsy”.1 Establishment of SPR analysis method for fusion gene based on improved nonlinear hybridization chain reactionThe nonlinear hybridization chain reaction（HCR）system is realized by kinetically controlled chain-branching growth of the DNA dendrimers from successive displacement reaction of double-stranded DNA substrates and assistant strands.The large molecular mass of nanostructure can significantly enhance the signal of surface plasmon resonance.Thus,in this study,a rapid SPR biosensing strategy has been developed for the detection of PML/RARαsubtypes from acute promyelocytic leukemia fusion gene based on efficient self-assembly of only three oligonucleotide stands on SPR interface,forming a sandwich structure of probe-target-dendrimer.This method not only obtains good analytical performance（LOD:“L”subtype:0.72 pM;“S”subtype:0.65 pM）,but also achieves simple and efficient analysis process.Moreover,the PCR products of different PML/RARαsubtypes are successfully identified.Owing to the SPR biosensor is more suitable for detection applications under different environmental conditions with the advantages of real-time,enzyme-free and label-free,this method provides a new potential application platform for fusion gene analysis and early diagnosis of related clinical diseases.2 Establishment of SPR analysis method for fusion gene based on aptamer-functionalized DNA hydrogelDNA self-assembly based on modular structure is an important form for development of multi-dimensional supramolecular nanostructures.Aptamer-functionalized DNA hydrogel not only has better sequence arrangement and larger molecular weight,but also can encapsulate macromolecular substances,leading to formation of supramolecular polymers.Therefore,two types of DNA self-assembled X polymers containing streptavidin（SA）aptamers are designed and prepared based on four nucleic acid chains.Then X1 and X2 are hybridized on the SPR interface to form DNA network hydrogel nanostructure.Finally,SA is encapsulated into hydrogel by the aptamers.Thus,the developed strategy achieves dramatic enhancement of the SPR signal.This method is applied to the detection of PML/RARα“S”subtype.A linear range of 100 fM to 10nM is obtained and the LOD is 45.22 fM,which breaks through the previous limitation of the lowest limit of linear range at pM level based on self-assembly SPR method.At the same time,this method improves the analysis performance of PCR products by an order of magnitude.Therefore,this study provides new experience for further exploring the self-assembly rule based on SPR interface and new idea for the construction of biosensing methods with better analytical performance.3 Establishment of fluorescence analysis method for fusion gene based on magnetic composite probe-actuated deblocking of stem-loop structureSecondary structures in long circulating tumor nucleic acids have potential obstacles to specific location point hybridization detection of this gene fragments.Exploration of biosensing strategies requires to selectively change the nucleic acid conformation and induce the signal switching.Here a self-assembled magnetic composite probe（MCP）is fabricated by hybridization reaction of Link DNA and“Y”-junction-DNA nanostructure on the surface of magnetic bead,contributing capture,stem-loop structure unlock and enrichment of PML/RARαDNA“L”subtype.Then,by integrating the MCP-actuated reactor,a one-step“off-on”signal switching MoS₂@FAM-probe biosensing method is developed for efficient detection of PML/RARαDNA“L”subtype.The proposed biosensor is capable of detecting 100 bases PML/RARαDNA“L”subtype with a wide linear range of 1 pM to 200 nM and limit of detection down to 0.223 pM without any signal amplification.In addition,the biosensing method is successfully applied for detection of target in the serum samples,which provides a basis for further research on the application of direct hybridization detection of nucleic acid gene fragments from ctDNA and ctRNA.