Construction And Application Of Fluorescence Sensor Based On DNA Templated Copper Nanoclusters

Copper nanoclusters refer to clusters that are composed of a few to dozens of copper atoms gathered into relatively stable clusters through physical and chemical binding forces.Compared with precious metal nanoclusters,copper has more abundant reserves on the earth,making copper nanoclusters easy to obtain and low cost.In addition,as an emerging fluorescent nanomaterial,copper nanoclusters have excellent properties such as rapid synthesis,ultra-small size,good biocompatibility,and environmental friendliness compared with traditional fluorescent materials,whitch makes copper nanoclusters have received more and more extensive research in metal ion detection,biosensing,fluorescence imaging and so on.In this paper,we used hairpin DNA and double-stranded DNA as templates to rapidly synthesize fluorescent copper nanoclusters.Based on different detection principles,new fluorescent sensor methods for detecting Chlamydia trachomatis target genes and folic acid was constructed.The first chapter of this article described the research background,research significance and research status of the target genes of C.trachomatis,folic acid and other biomolecules.Then we introduced the principle of fluorescence phenomenon,the sensing principle and response mechanism of fluorescence sensor,and the superior performance of biosensor.Finally,we focused on the synthesis of new fluorescent copper nanocluster materials,the advantages of DNA as a synthesis template,and the detection application of copper nanoclusters in biosensing.The second chapter of this paper designed a method for synthesizing copper nanoclusters using hairpin DNA as a template.The synthesized copper nanoclusters were quasi-spherical with an average particle size of2.12 nm.Based on the principle that the conformation of the hairpin DNA will be changed in the presence of C.trachomatis target gene,a fast,sensitive,and highly specific fluorescent biosensor for the detection of C.trachomatis target sequence was established.The sequences of other pathogens did not significantly interfere with the detection of C.trachomatis target sequences.The fluorescence response signal was linearly correlated with the target gene content of C.trachomatis in the range of 5.0×10^-8～9.5×10^-7 mol/L,the correlation coefficient was 0.9939,and the detection limit was 18.5×10^-9mol/L.The fluorescent biosensor had been successfully applied to the detection of C.trachomatis target sequence in He La cell lysate,providing a new idea for the detection of C.trachomatis.In Chapter 3,a copper nanocluster with high fluorescence intensity was synthesized using copper sulfate as the precursor,sodium ascorbate as the reducing agent,and double-stranded DNA as the template.We adopted a"bottom-up"method to synthesize copper nanoclusters,and used ultraviolet-visible spectrophotometer（UV-Vis）,Fluorescence Spectrophotometer（FL）,Transmission Electron Microscopy（TEM）,Fourier Infrared Spectroscopy（FT-IR）,X-ray Photoelectron Spectroscopy（XPS）and other technical methods to characterize the properties of copper nanoclusters.The mechanism of folic acid quenching the fluorescence of copper nanoclusters was discussed.The fluorescence quenching efficiency of copper nanoclusters increased with the increase of reaction temperature.Therefore,we proposed a possible dynamic quenching mechanism.At the same time,we found that the ultraviolet absorption peak of folic acid at 345 nm overlaps with the excitation spectrum of copper nanoclusters.Folic acid could selectively quench the fluorescence of copper nanoclusters based on the internal filtration effect.Based on the above principles,we constructed a new type of fluorescence sensor for fast and sensitive detection of folic acid.Under the best experimental conditions explored,the concentration of folic acid was linearly related to the fluorescence change value（△F/F₀）in the range of1.25～50×10^-6 mol/L,with a detection limit of 0.34×10^-6 mol/L.The fluorescence sensor was actually applied to the detection of folic acid in serum and medicines,and a good recovery rate of standard addition was obtained,indicating that the method has the possibility of practical application.