Study On The Novel Electrochemical Active-inactive Switch MB Biosensor Based On Magnetic Nanoparticles And Its Application

DNA(Deoxyribonucleic acid) is the hereditary material in humans and almost all other organisms,with the functions in the storage and transmission of hereditary information.The normal operation of the various functions in life is closely related to DNA.Little changes in the DNA sequence,such as base-pair mutation,error,loss and increment,will affect the normality of the germ plasm and lead to the appearance of the diseases.So the separation and analysis of DNA are playing an increasingly important role in the medical diagnosis,pharmaceutical research,bioengineering and other fields.Compared to the traditional sequence detection methods,DNA biosensor, a new conception of genome detection based on Watson-Crick base match principle, is of great advantages such as easy handling,quick detection and favorable selectivity and no pollution.Nowadays it has become one of the most important methods in the DNA detection.The research in the combination of the DNA biosensor with electrochemistry,which is high-sensitive,has been one of the most attractive fields in genome study.Nano-materials,with the reputation of "the most promising material in the 21th century",are considered the hot area in the material research.Their unique physical and chemical properties offer excellent prospects for the biosensor.The nanoparticles applied in the biosensor,will enhance its sensitivity and selectivity effectively.Molecular beacon was designed firstly by Tyagi and Kramer in 1996.It has the advantages of high selectivity and specificity in the conversion between its stem-loop structure and stretch structure.Compared with the traditional molecular beacons based on the principle of fluorescence resonance energy transfer,the electrochemical molecular beacons are more selective and easy-operating,and have been applied in many application scopes.To date,molecular beacons have been widely used in real-time monitoring of PCR,the rapid mutation analysis,dynamic analysis of living cells and DNA -protein interaction research.The goal of our study is to design a novel electrochemical active/inactive switch MB biosensor,which combines nano-technology,molecular beacon and electrochemical technology.The biosensor is integrated by the accumulation of magnetic nanoparticles,the high selectivity and specificity of electrochemical molecular beacons and the simplicity of electrochemical biosensor.It will detect the target effectively and quickly,not only broadening the application of MB but also providing new method for the development of the biosensor.The dissertation includes three chapters:Chapter 1 PrefaceDNA is the hereditary material of the life.The normal operation of the various functions in life is closely related to DNA.Because of the high selectivity and specificity,moleculat beacons have been studied widely.In this chapter,we made a brief introduction of the DNA,molecular beacons and magnetic nanoparticles.It involved the basic principles,the classification and the development,which provided the theoretical principle for the paper.At last the purpose and innovation of the dissertation were pointed out.Chapter 2 Synthesis and characterization of the novel core-shell magnetic nanoparticlesMagnetic nanoparticles,due to their unique properties,have a wide range of applications in the biological separation,medical diagnosis and so on.In this chapter, we first synthesized Fe₃O₄,Fe₃O₄@Au and Fe₃O₄@SiO₂ nanoparticles in different solutions,analyzed the effects of the experiment conditions and optimized the synthesis methods.Then,their structures were characterized by transmission electron microscopy(TEM),scanning electron microscopy(SEM),ultraviolet-visible spectroscopy(UV-Vis) and X-ray diffraction(XRD),indicating that the nanoparticles possessed excellent dispersity and well stability,which showed potential application value in the field of Biochemistry.Chapter 3 Study of the novel electrical active-inactive DNA biosensor based on Fe₃O₄@Au nanoparticlsp53 gene is an important tumor suppressor gene.It is found that 50%human tumors link to p53 gene loss or mutation.So it is of great importance to analyse and detect p53 gene.In this chapter a novel electrical active-inactive switch molecular beacon was synthesized first.Then one kind of DNA electrochemical biosensor with sandwich structure was constructed based on the combination of the molecular beacon and Fe₃O₄@Au nanoparticls.Applying the biosensor in the analysis and detection of p53 target showed that the detective limit of the biosensor was 1.1×10^-10mol/L and the identification rate of detecting the single-base mismatch reached 80%over.It has a potential application in biomedicine and so on.