Study On The Chemiluminescent Catalytic Activity Of Magnetic Nanoparticles Mediated By K₄Fe（CN）₆ And Its Applications

The CL analysis method is a quantitative analysis method based on the CL intensity and has the advantages of high sensitivity,wide linear range,and simple instrumentation.Immunoassay is an analytical method based on the specific recognition between antigen and antibody.Chemiluminescent immunoassay（CLIA）combines the high sensitivity of CL with the good specificity of immunoassays.Natural enzymes are commonly used as labels in CLIA.However,natural enzymes suffer from the high price,complicated labeling processes,and poor stability.Therefore,the search for materials with peroxidase-like activity to replace natural enzymes has been becoming a research hotspot in recent years.In 2007,Yan’s group reported the peroxidase-like activity of magnetic nanoparticles（MNPs）.They found that MNPs could catalyze the color reaction of3,3’,5,5’-tetramethylbenzidine（TMB）and o-phenylenediamine（OPD）in the presence of H₂O₂.Since then,the researches of the mimetic peroxidase activity of nanomaterials emerged prominently.Various nanomaterials including gold nanoparticles,silver nanoparticles,graphene oxide,carbon dots,zinc oxide nanoparticles and so on have been reported to possess the peroxidase-like activity and are capable of catalyzing the luminol CL reaction.These nanomaterials have also been applied to the detection of glucose,tumor markers,uric acid,glutathione and so on.Studies showed that the catalytic activity of nanomaterials was affected by factors such as size and surface properties.Some nanomaterials with good catalytic activity encountered the problem of inhibited catalytic activity after being modified with bio-recognition molecules,which limits their applications as a label in CLIA.In the present study,we developed a novel CLIA method based on an in-situ generation of Prussian Blue（PB）strategy.Herein,the biological-recognition molecules were modified on the surface of MNPs,and the MNPs were used as a label in the CLIA.After the immunoreaction,K₄Fe（CN）₆ was added into the wells of the96-well plate for the in-situ generation of PB.The formed PB was capable of catalyzing the luminol CL reaction.The analytes could be quantitatively determined based on the CL intensity.The thesis includes three parts.Firstly,the kinetic curves of K₄Fe（CN）₆-mediated luminol-MNPs CL reaction were investigated.In the absence of MNPs,K₄Fe（CN）₆-luminol reaction showed a very weak CL intensity indicating that K₄Fe（CN）₆ could not catalyze luminol to emit light.However,In the presence of MNPs,the CL intensity of the K₄Fe（CN）₆-luminol system was highly enhanced.The mechanism studies indicated that PB was generated during the incubation of K₄Fe（CN）₆ with MNPs,which was confirmed by using UV-Vis（Ultraviolet Visible Spectra,UV-Vis）,FT-IR（Fourier Transform Infrared Spectra,FT-IR）,and TEM（Transmission Electron Microscope,TEM）.Free radicals quenching experiments showed that O₂^·-,·OH and ¹O₂ were involved in the oxidation of luminol.Then,we optimized the experimental conditions including the concentration of K₄Fe（CN）₆ and luminol,the reaction time between K₄Fe（CN）₆ and MNPs,the p H of luminol,and the reaction environment.The optimized experimental conditions are as follows.The concentration of K₄Fe（CN）₆ and luminol were 50μM and 100μM,respectively.The reaction time was 40 min and the reaction environment was 5 m M HCl.The p H of the dilution solution for luminol was at 12.Under the optimized experimental conditions,we investigated the catalytic ability of COOH-MNPs and SA-MNPs.In the range of 0.0046-0.75μg m L^-1,it showed good linear relationship between the concentration of MNPs and CL intensity.The assay performance showed that after modification,the SA-MNPs still exhibited good catalytic ability.In chapter 3,we applied the proposed CL reaction to immunoassay for the detection of rabbit Ig G（r Ig G）as a model analyte.SA-MNPs was chosen as a label in the CLIA.The CL intensity increased linearly with the concentration of r Ig G ranging from 0.625-20 ng m L^-1.The linear regression equation was I=0.0811 C+0.333.The LOD was 0.59 ng m L^-1.The proposed CLIA method was also compared with the traditional ELISA（Enzyme-Linked Immunosorbent Assay,ELISA）method by using the HRP（Horseradish Peroxidase,HRP）-catalyzed OPD colorimetric method.The LOD of the ELISA method was 0.9 ng m L^-1.The results demonstrated that the proposed CLIA method was comparable to the ELISA method.Then,we also applied the in-situ generation of PB strategy for the detection of CEA（Carcino-Embryonic Antigen,CEA）.The linear range for CEA was from 0.5 to 100 ng m L^-1 and the LOD was 0.28 ng m L^-1.In chapter 4,the in-situ generation of PB strategy was extended to the detection of sequence-specific DNA related to the Hepatitis B virus（HBV）.The linear range for the detection of sequence-specific DNA was from 0.065 to 1.0 pmol and the LOD was0.044 pmol.In summary,we used the in-situ generation of PB as a strategy to establish the K₄Fe（CN）₆-mediated luminol-MNPs CL reaction.Then SA-MNPs was used as a label in CLIA method,and successfully detected r Ig G and CEA.The proposed CLIA method was also extended to the detection of sequence-specific DNA molecules.