Preparation Of Carbon Dots Fluorescence Resonance Energy Donor And Its Application In The Analysis Of Biomarkers

Biomarkers,including proteins,nucleic acids and enzymes,are the "molecular signature" of the physiological state of disease at a specific time.The abnormal expression of biomarkers in tumor tissues or serum is greatly related to the existence of some diseases.Therefore,it is very important to design a simple,rapid and accurate detection method for the detection of biomarkers,which is benefit for the early diagnosis and treatment of disease.Fluorescence method is widely used in biomolecular analysis because of its simple quantitative process,easy detection of samples,time-saving and high sensitivity.Furthermore,the analytical method based on fluorescence resonance energy transfer(FRET)has also been widely applied in analytical chemistry and biological imaging because of its simplicity,high efficiency,low background interference and high accuracy.As a new type of carbon nanomaterials,carbon dots(CDs)have become ideal energy donors for FRET owing to the advantages of simple preparation,good water solubility,good photostability and adjustable light-emitting wavelength.At present,the establish of a series of simple,rapid and sensitive FRET analytical method by using CDs as energy donor after surface modification is significant for expanding the application of CDs-based FRET on the analysis of biomarkers.Therefore,the main contents of this thesis include the following three parts:1.The preparation of CDs rich in carboxyl groups as energy donors for FRET.In the process of preparing CDs,some groups of raw material molecules will remain on the surface of CDs.Therefore,citric acid rich in carboxyl group is chosen as carbon source and spermine is used as nitrogen source to provide good optical properties.Carboxyl-rich CDs are prepared by simple hydrothermal method.The results of X-ray photoelectron spectroscopy(XPS)and Fourier transform infrared spectroscopy(FT-IR)showed that the surface of the CDs was rich in carboxyl functional groups,and the CDs had excellent light stability,salt stability and oxidation resistance.The rich carboxyl functional groups on the surface of CDs and excellent optical properties provide a potential possibility for the subsequent modification of CDs and the use of CDs as energy donors for FRET analysis.2.We have developed a universal detection platform for cancer biomarkers prostate specific antigen(PSA)by combining CDs-based FRET analytical method and enzymatic catalyzed hairpin assembly(CHA)strategy.The design included DNA(T),blocker DNA(B),hairpin DNA(H1),DNA(H3)and amino modified hairpin DNA(H2).By coupling amino-modified hairpin DNA(H2)on carboxyl-rich CDs.In the absence of PSA,the hairpin DNA is adsorbed by graphene oxide(GO),and the fluorescence of CDs is quenched by GO through FRET.In the presence of PSA,CHA can be triggered and formed "Y" double-stranded DNA structure.The fluorescence intensity of CDs gradually recover with the increasing concentration of PSA.The signal ratio of fluorescence intensity has a good linear relationship with the concentration of PSA in the range of 1-100 ng/mL,with the the limit of detection(LOD)of 0.22 ng/mL(3?/k).In addition,by properly changing the corresponding aptamer sequence,this method can also realize the detection of other targets,such as carcinoembryonic antigen(CEA)and adenosine triphosphate(ATP),with the LOD of 0.56 ng/mL(3?/k)and 80 nM(3?/k),respectively.Furthermore,this method has excellent selectivity,and the establishment of a universal sensing platform is of great significance for early diagnosis and treatment of clinical diseases.3.We have established a FRET analytical method with dual fluoresence emission under a single excitation wavelength for simultaneous detection of dual-target miRNA by using CDs and quantum dots(QDs)as energy donors and combining hybridization chain reaction(HCR)strategy.The design included hairpin DNA(H1),DNA(H3),amino modified hairpin DNA(H2)and biotin-modified hairpin DNA(H4).Amino-modified hairpin DNA(H2)was coupled on carboxyl-rich CDs and biotin-modified hairpin DNA(H4)was coupled on streptavidin-modified QDs.In the absence of miRNA-141 and miRNA-21,the hairpin DNA is adsorbed by GO,and the fluorescence of CDs and QDs are quenched by GO through FRET.In the presence of miRNA-141,the HCR between DNA(H1)and CDs-DNA(H2)will be triggered;In the presence of miRNA-21,the HCR between DNA(H3)and QDs-DNA(H4)will be triggered.The fluorescence intensity of CDs and QDs gradually recover.The fluorescence intensity signal ratio of CDs and QDs has a good linear relationship with the concentration of miRNA-141 and miRNA-21,respectively.The linear range is 0.1-10 nM,with the LOD of 50 pM(3?/k)and 60 pM(3?/k),respectively.Compared with single biomarker detection,double target detection can avoid false positive results and improve the accuracy of disease diagnosis.In conclusion,by taking carboxyl-rich CDs as energy donors,a series of FRET analytical method has been established with the combination of self-assembly of DNA circuits strategy for rapid,sensitive and accurate detection of biomarkers.At the same time,these methods supply a universal detection platform for the analysis biomarkers and can be used for multiple targets detection simultaneously,which greatly improve the sensitivity and accuracy of diagnosis.Overall,the established methods with the advantages of simple,low cost,high sensitivity and accuracy provide an effective strategy for early diagnosis of disease,which is of great significance for clinical application.