Research And Application On Novel Biosensors For Disease Markers

Detection of disease markers can supply observation data for the diagnosis and disease's curative effect monitoring and prognosis.Recently,along with the development of molecular biotechnology,the development of simple,convenient,fast,sensitive,well practice biosensor for disease markers detection has become more and more important.In this thesis,some new methods(based on fluorescence,colorimetric,and ultraviolet)for disease markers monitoring has been developed.This paper includes four chapters.In the first chapter,a general introduction of disease markers was made,including their definition,features,categories,clinical significant and some other points.Then,the principles of biosensor,relevant detection technologies and the application in disease markers were also introduced.The purpose and signification of this thesis were described.In the second chapter,based on the protecting effect of folate receptor(FR)toward folic acid(FA)modified DNA and the signal amplification of hybridization chain reaction,a sensitive fluorescent strategy for the detection of folate receptors(FRs)had been proposed.In the present of the target(FR),the specific folic acid folate receptor(FA-FR)interaction can protect the DNA from enzymolysis by exonuclease I(Exo I).Then,FA was modified with one single strand DNA,which can initiate the hybridization of the other two single strand DNAs to form a DNA polymer(long double strand DNA)through isothermal hybridization chain reaction(HCR).Since SYBR Green I can bind the minor groove of dsDNA to emit strong fluorescence signal,so high fluorescence intensity can be detected.Based on which,a sensitive fluorescence biosensor can be developed for FR detection.The enhanced fluorescence intensity has a linear relationship with the FR concentration from 10 pM to 10 nM and a detection limit of 3.3 pM was achieved.The proposed method had been applied to detect FR in cells samples with satisfied results.In the third chapter,specific nucleic acid detection by using simple and low-cost assays is important in modern medicine and chemistry.But most current methods for the detection of low concentrations of DNA need costly and sophisticated instruments.So we develop a simple DNA detection platform based on gold nanorods etching process,in which the size and shape of the gold nanorods with surface plasmon resonance shift.Through the combination of isothermal hybridization chain reaction,we can amplifying the signal and detect lower concentrations of nucleic acids.Tt is expected to a great interest in modern medicine and chemistry owing to the high sensitivity and selectivity of this DNA assay.In the fourth chapter,a novel fluorescent biosensor was constructed based on the adsorption of the nucleic acid backbone to some nano materials.Iron oxide nanoparticles adsorb fluorescently labeled DNA oligonucleotides via the backbone phosphate and quench the fluorescence.Zinc ions in alkaline phosphatase protein cavity can attract with the phosphate groups of DNA skeleton and the 5'ends of DNA were phosphorylated by alkaline phosphatase compete phosphate from the DNA backbone and inorganic phosphate displaces adsorbed DNA to increase fluorescence.The fluorescence intensity has a linear relationship with the ALP concentration from 0.1U to 50U.This is a new way of building a fluorescence biosensorusing relies on DNA desorption from iron oxide nanoparticles.