Study On The Fluorescence Properties Of DNA-stabilized Copper Nanoclusters And Their Analytical Applications

As a new type of fluorescent materials,the application of metal nanoclusters is of great concern in the field of luminescence analysis and biosensing in recent years.Copper nanoclusters(CuNCs)have broad application prospects in the field of fluorescence sensing because of their low cost,easy synthesis and good biocompatibility.However,there are some problems such as low fluorescence quantum efficiency and poor stability of copper nanoclusters as fluorescent probes.In this thesis,DNA-stabilized copper nanoclusters were used as research objects to explore the method to improve the fluorescence properties of these probes,and to construct a new biosensor method.The details are as follows:1.Cu nanoclusters-based nanosensor for detection of streptavidinIn this study,biotin was labeled at the 3' end of the poly-AT double-stranded DNA,and when the target molecule streptavidin was added to the system,it was possible to specifically bind to biotin,which inhibited the exonuclease ? hydrolysis.When Cu2+and ascorbic acid were added into the system,copper nanoclusters with double-stranded DNA as template were formed,which showed strong fluorescence signal.On the other hand,double-stranded DNA template was hydrolyzed by exonuclease ? and could not form copper nanoclusters.Thus,the concentration of streptavidin can be determined by the change in fluorescence intensity of the system.This method is simple,low cost,high sensitivity and good selectivity.It provides a general fluorescent method for detecting similar biotin and streptavidin model recognition,and is expected to be used to detect interaction between other types of small molecules and target proteins.2.Study on the stability of DNA-templated Cu nanoclusters The application in the field of bioanalysis of copper nanoclusters(DNA-CuNCs)synthesized with double-stranded DNA as templates have been hindered by their poor stability.In this study,we investigated the fluorescence stability of the DNA-CuNCs by a series of experiments,such as adding free radical scavenger,nitrogen,oxygen.It found that the reason for its poor stability is mainly due to the attack of hydroxyl radicals and the reason of oxygen oxidation.Finally,the stability of DNA-CuNCs was extended from tens of minutes to twenty days by reducing the contact area with oxygen.It provides favorable conditions for the further study of copper nanoclusters and is expected to be used to expand the application of fluorescent copper nanoclusters.3.Histone-DNA interactions:a approach to improve the fluorescence performances of DNA-templated Cu nanoclustersIn biology,histones are highly alkaline proteins in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes.In this study,it was found that the addition of histone could increase the fluorescence quantum efficiency of DNA-CuNCs from 2%to 14.6%in the process of synthesizing copper nanoclusters with double-stranded DNA as template.We investigated the effect of histone on the fluorescence intensity of DNA-CuNCs by means of Circular Dichroism spectroscopy,Photoelectron spectroscopy,and UV-Visible absorption spectroscopy.In addition,histones can also significantly improve the fluorescence stability of DNA-CuNCs.Finally,we have shown here that histone-DNA binding events can also be used to greatly improve the fluorescence intensity and photostability of DNA-CuNCs.The probe was used for the detection of sulfur ion and TDT activity.This study provides a new method for improving the fluorescence properties of CuNCs in order to establish a highly sensitive method for fluorescence sensing.4.A fluorescence biosensor based on Photoelectron Induced Transfer for ultrasensitive detection of exonuclease ? activityIn this study,DNA consisting of several CG base sequence with fluorescein at the 3' end was designed and can be self-assembled into a double-stranded DNA in a buffer solution.The probe fluorescence is extinguished because of Photoelectron Induced Transfer between G-base and fluorescein.The exonuclease ? can hydrolyze the double-stranded DNA in the direction of 3'?5' to form the single nucleotide,so the fluorescent molecule are free to the solution.The fluorescein is restored and the fluorescence intensity showed a good linear relationship in the concentration range 5×10-4-5 U/ML exonuclease ?.The method is simple,rapid and sensitive,and can be used for the screening of exonuclease ? inhibitors and the qualitative analysis of exonuclease III in HaLa cells.