The Study Of Fluorescent Bioanalysis Strategies With Nucleic Acid Amplification Technology On Nanocarrier

Fluorescence biosensor technology is an analytical tool that combines fluorescence analysis method with biomolecules(such as nucleic acid,protein and small molecules)to form biometric identification elements,which could react with targets to produce fluorescence signals to achieve qualitative and quantitative detection.In recent years,DNA nanomachines developed by the combination of fluorescent biosensor technology and DNA nanostructures have become a research hotspot in the fields of biological assay,clinical diagnosis,tumor drug delivery and pathogenic microorganism exploration due to their advantages of simple operation,high efficiency and high stability.However,traditional DNA nanomachines were limited by complex operation,low loading efficiency and execution efficiency,which limited the sensitive detection of biomarkers with low abundance.Therefore,the introduction of nucleic acid amplification strategy could cascade amplify the signal to improve the accuracy and sensitivity of analysis detection methods.Herein,we constructed novel DNA nanomachines with nucleic acid amplification technology on nanocarrier for sensitive detection of microRNAs.The specific works are as follows:1.A Double-Loop Shaped 3D DNA Nanomachine and Its Application for Sensitive Fluorescent Bioassay of MicroRNAThe traditional DNA tweezers were normally constructed by self-assembly of three single-stranded DNAs,which regulated the open and closed states on the basis of linear conformational changes.However,the linear DNA nanostructure lacked the necessary rigidity to maintain the open structure of the tweezers,resulting in higher background signal generation.In order to overcome this defect,we designed a double-loop shaped3D DNA nanomachine with high mechanical rigidity,which was constructed by mechanical interlocking double-DNA-ring with two single-stranded DNAs.With the help of the target microRNA-21,the 3D DNA nanomachine could achieve open-close structural changes in three-dimensional space,increasing the adjustable distance range effectively.Compared with linear track,double-loop tracks with higher mechanical rigidity could reduce the background signal.The experimental results demonstrated that the 3D DNA nanomachines could be used for quantitative analysis of microRNA-21based on fluorescence signals generated by three-dimensional structural transformation with the detection range of 1-100 nmol/L and the LOD of 0.3 nmol/L.This work provided a new avenue for the construction of novel DNA nanostructures,and provided an effective tool for biomolecule diagnosis and biomedical detection.2.A Target-initiated Autocatalytic 3D DNA Nanomachine for High-Efficiently Amplified Detection of MicroRNAThe nucleic acid amplification strategy combined with the traditional 3D DNA nanomachines was unidirectional or a single repeating cycle,which limited the execution efficiency and movement processivity of the nanomachines.Therefore,a novel autocatalytic three-dimensional(3D)DNA nanomachine was constructed based on cross-catalytic hairpin assembly on the gold nanoparticles(Au NPs)to generate self-powered efficient cyclic amplification.Firstly,four DNA hairpins were modified on Au NPs.In presence of target microRNA-203a,the 3D DNA nanomachines were triggered to activate a series of CHA reactions,in which,based on the reasonable design,the product of the first CHA reaction was the trigger of the second CHA reaction and the product of the second CHA reaction was the trigger of the first reaction,generating an efficient self-powered CHA amplification strategy without adding fuel DNA strands or protein enzymes externally.Due to high-efficiently recycling utilization of CHA products,the proposed autocatalytic 3D DNA nanomachine maximally improved amplification efficiency to produce high-efficiency fluorescence signal amplification.The experimental results demonstrated that the autocatalytic 3D DNA nanomachine has good analytical performances for the detection of microRNA-21 in vitro and in living cells.The linear range was 0.1-150 nmol/L with the LOD of 74 pmol/L.This work proposed a new idea to construct highly processive DNA nanomachines,which exhibited promising potential for ultrasensitive bioanalysis and offered a new avenue for great application in biosensing and early clinical diagnosis of diseases.3.An Amphiphilic 3D DNA Nanomachine based on Hydrophobic-Driven Self-Assembly for High Efficiency Fluorescence Detection of MicroRNAThe contradiction between the effective immobilization of bioactive substances and their reaction efficiency is still one of the important challenges in the field of chemistry.The traditional 3D DNA nanomachines usually immobilized DNA reaction substrates on hard interfaces such as gold nanoparticles or silica microspheres by covalent bond interaction,which limited the free diffusion of substrates and thus reduced the reactivity of substrates.Therefore,we constructed an amphiphilic 3D DNA nanomachine by hydrophobic driven self-assembly of amphiphilic oligonucleotides.The amphiphilic oligonucleotides were formed by modifying hydrophobic groups at the ends of oligonucleotides.In aqueous solution,the amphiphilic DNA nanospheres were formed by hydrophobic driven self-assembly of amphiphilic oligonucleotides.The DNA substrates on the surface of the nanosphere could trigger the CHA cycle by binding to the target to realize the walking amplification process of DNA Walker.Compared with the traditional homogeneous reaction system,the local concentration of the substrates was increased by at least 9.0×10~5 times by the self-assembly based on hydrophobic interaction.And the hydrophobic core,as a flexible carrier,made the DNA substrates move freely on the surface of amphiphilic DNA nanosphere,thus effectively improving the reaction activity and reaction efficiency.With microRNA-155 as a model,the detection range was 0.5 to 100 nmol/L with a limit of detection(LOD)of 274 pmol/L,and the reaction time was reduced to 30 min.In addition,the amphiphile 3D DNA nanomachines have demonstrated good analytical performances in intracellular imaging.This method could be easily expanded for the bioassay of various biomarkers,such as nucleotides,proteins and cells,offering a new avenue for simple and efficient applications in bioanalysis and clinical diagnosis.