Study On Asymmetric Porous Nanosensors With Ternary Structure Toward High-sensitivity MiRNA Detection

Micro RNAs(miRNAs)are promising markers for tumor screening and diagnosis in clinical diagnosis and treatment.The development of simple,rapid,sensitive and specific miRNA detection methods is of great significance to promote the early and high sensitivity detection of tumor marker miRNA.Fluorescent biosensors based on nanomaterials have been widely used in the detection of biomarkers.The detection of miRNA based on nucleic acid aptamer technology and nano fluorescence quencher has great potential in the early and rapid detection of tumor markers.However,most of the nanoquenchers have a limited external surface area,which limits the amount of fluorescent aptamers that can be carried by the particles.At present,most of the detection systems use DNA enzyme in the liquid phase simultaneously,which may block the pores of nanoquencher and interfere with the detection of fluorescence intensity.In addition,the reaction conditions of free enzyme are strict and easy to inactivate.The shortcomings of the above detection system limit the design of "smart" nanosensors to some extent.Therefore,how to overcome the shortcomings of the existing detection system is worth studying.In this paper,we combined the advantages of nanomaterials,aptamers and exonuclease assisted signal amplification technology to construct a ternary asymmetric nanosensor based on mesoporous dopamine(MPDA)-Au nanoparticles-DNA exonuclease for the detection of high sensitivity miRNA in biological system.As an excellent fluorescence quencher,PDA nanoparticles can form a mesoporous structure to protect the adsorbed single-stranded DNA probes.In the choice of enzyme immobilization module,the surface modification methods of gold nanoparticles and PDA are mature,and the surface modification based on Au-S coordination bond is conducive to the realization of selective DNA enzyme coupling.Specifically,the ternary asymmetric nanosensor is composed of MPDA-Au-enzyme.Among them,the MPDA side serves as the "sensing unit" to guide the fluorescence recovery controlled by target miRNA binding and heteroduplex release,and the Au side is the "amplification unit" for the target cycle of immobilized enzyme cleavage.The main research contents are as follows:(1)Controllable preparation of Janus MPDA-Au nanoparticles and exploration of the asymmetric recombination law in nanoscaleFirstly,the morphology and structure of MPDA nanoparticles were studied.MPDA nanoparticles(about 200 nm)with pore structure were synthesized by using surfactant(Pluronic(?)F127,F127)as the template.By further controlling the polymerization rate of PDA,adjusting the ratio of reactants,adding rate and other factors to control the particle and pore structure,the MPDA particles with rich pore structure and size of about 100 nm were obtained.Then,the morphology of Janus particles is studied.Janus MPDA-Au was prepared by electrostatic repulsion.Based on the chelation and reduction of catechol groups on metal ions,gold nanoparticles were grown asymmetrically,and Janus MPDA-Au nanoparticles composed of MPDA part(average diameter about 200 nm)and Au part(about 30 nm)were successfully constructed.In addition,the influencing factors and control laws of the selective growth of gold nanoparticles on the surface of MPDA were explored,and a series of Janus nanoparticles with adjustable gold particle size were obtained.In addition,according to the Pickering emulsion-mediated particle surface growth method,part of the surface of the host MPDA nanoparticles was protected,and the selective growth of the guest Au particles was achieved.Janus MPDA-Au nanoparticles composed of MPDA part(about100 nm)and Au part(about 30 nm)were successfully constructed.This provides new ideas for the design of nanomaterials.(2)Construction of MPDA-Au-enzyme ternary asymmetric nanosensor and miRNA detection applicationBased on the asymmetric morphology of MPDA-Au,the special binding DNA enzyme was modified on the surface of gold particles by the specific binding of gold and disulfide ligands.Finally,the MPDA-Au-enzyme ternary asymmetric nanosensor with integrated aptamer carrying,miRNA recognition response,circulation,and signal amplification functions was obtained.In addition,the fluorescence quenching effect of Janus nanoquencher,the cooperative signal amplification based on asymmetric enzyme modification and the performance evaluation of miRNA detection were studied.By experimental analysis,the detection limit of the nanodetector was 32.0 f M,which has excellent selection specificity.In addition,the sensor had the feasibility of detecting trace amounts of target miRNA in complex biological samples.In summary,this paper successfully constructed a ternary asymmetric nanosensor based on MPDA-Au-enzyme.The system realized the fine regulation of the morphology of asymmetric nanomaterials,integrated functional modules in one material construction,and showed high sensitivity and specificity in the field of miRNA detection.This provides a new strategy for the construction of composite materials based on nanoquencher,and also provides a new idea and new model with reference value for the development of high-performance miRNA nano fluorescence detectors.