The Research On The Novel Fluorescence Analysis Methods Based On The Utilization Of Dna As Recognition Units

Biosensor is an analysis device that combines a biometric element and a physicochemical detector,which can be used to detect biochemical substances and convert their concentration and other physical and chemical information into qualitative and quantitative detection signals.Since biosensor has many advantages such as easy operation,rapidity,good specificity,low cost and high sensitivity,it is widely used in biochemical analysis,food and drug safety analysis,clinical diagnostic analysis and environmental monitoring.Nucleic acid is not only the carrier of biological genetic information,but also has biological functions such as catalytic and specific recognition.Functional nucleic acids have the advantages of high specificity,good biocompatibility,good stability,easy synthesis and modification,and high sensitivity.They have been widely used in biological analysis.This research focus on novel fluorescence analysis methods based on the utilization of DNA as recognition units.We have further developed some new fluorescence sensing system combining the nucleic acid dye,Tb³⁺and nanoparticles.Our purposed methods have been used for the rapid detection of hydrogen peroxide?H₂O₂?and microRNA with high sensitivity and good selectivity,which provides a new thought and method in the field of biological sensing.This article was divided into four chapters as follows:Chapter 1:OverviewIn this chapter,the constitution and application of biosensors are firstly introduced and nucleic acid sensor is discussed emphatically.Then,the development of fluorescent assay is summarized.Finally,the research ideas and content of this paper are expounded.Chapter 2:Design and application research of label-free ratiometric fluorescent probe based on GelRed/[G₃T]₅/Tb³⁺In this work,we focus on developing a novel label-free ratiometric fluorescent assay for H₂O₂ by using GelRed/[G₃T]₅/Tb³⁺hybrid.According to our previous studies,an optimum single-stranded DNA?ssDNA?antenna ligand?[G₃T]₅?was identified for effective sensitization of the fluorescence of Tb³⁺.In our proposed hybrid,Tb³⁺was sensitized by[G₃T]₅?i.e.GelRed/[G₃T]₅?as a sensitive response signal with green fluorescence while GelRed intercalated into[G₃T]₅(i.e.[G₃T]₅/Tb³⁺)acts as a stable build-in reference with red emission.Since Hg²⁺can specifically interact with thymine?T?bases to form strong and stable T-Hg²⁺-T complexes,the green fluorescence of[G₃T]₅/Tb³⁺can be quenched.Moreover,with the addition of Cys,Hg²⁺is captured through the thiol group of Cys.The oxidation of Cys to cystine by H₂O₂ reverses the Cys-mediated fluorescence changes of[G₃T]₅/Tb³⁺,which promises a simple,cost-effective,sensitive and visual detection of H₂O₂.Additionally,the sensing strategy can be further extended to the monitoring of H₂O₂-produced oxidase-related reactions,such as GOx-biocatalyzed oxidation of glucose and the?AChE/ChOx?cascade biocatalytic processes.More significantly,our proposed method has been successfully integrated with a smartphone to make it useful for in-field fast measurement and realtime online analysis.Chapter 3:Preparation and character research of FITC@SiO₂In this study,FITC-doped silica nanoparticles?FITC@SiO₂?were prepared by a typical St?ber-based synthesis method.FITC@SiO₂ have a significant coffee ring phenomenon:upon evaporation under ambient conditions,as the three-phase contact line of the droplet in pinned on the surface due to either physical roughness or chemical heterogeneities,the evaporation process induces a capillary flow within the droplet which drives the suspended FITC@SiO₂ particles toward the contact line and leaves a ring-shaped structure at the rim.By investigating the influence of silicon nanoparticle concentration,glass matrix,droplet volume,ambient temperature,ionic strength,surfactant and surface groups on its coffee ring effect,suitable loop conditions were screened.Combined with coffee ring effect,DNA functionalized FITC@SiO₂ will have widespread application in the field of biological analysis.Chapter 4:Analysis method design and application research based on DNA functionalized FITC@SiO₂ fluorescent materials and coffee ring effectIn this work,we for the first time developed a visual and ratiometric fluorescent assay for the detection of microRNA based on Probe1-FITC@SiO₂ and Probe 2-MB.Combined with coffee ring effect,the fluorescence intensity of the ring is used as signal output.Besides,ratiometric fluorescent ring analysis method was established by using Rhodamine@SiO₂ as the internal reference to achieve visual detection of microRNA.