| Enzyme-linked immunosorbent assay(ELISA)is a very important analytical method.Nevertheless,in conventional ELISA,the preparation process of enzyme-labeled antibody is not only tedious,time-consuming,but also easy to lose enzyme activity,low enzyme labeling,which resulted in low detection sensitivity.Therefore,it is particularly important to design a solid carrier,which can load enzyme simply,stably and efficiently,for ELISA enzyme labeling.A cancer biomarker is a substance that can be objectively measured and evaluated as a biochemical indicator of cancer in the process of pathology or treatment to monitor the progress,regression and recurrence of the disease.Currently,many promising ELISA methods have been developed to detect cancer biomarkers.With the development of nanotechnology,bionanocomposites have been successfully combined with ELISA to amplify detection signals and improve sensitivity and accuracy.Metal-organic framework materials(MOFs)are one of the most promising materials for their high specific surface area,porosity,stability and adjustable size.Catalysis is one of the most attractive and rapidly developing fields in various applications of MOFs,which is mainly based on two ways:One is that the active site of the catalytic reaction is directly provided by the metal node and/or the organic ligand of MOFs.Another is that the guest material with catalytic activity is introduced into the holes of MOFs to form the MOFs-based composite catalyst.In this thesis,nanoMOFs with micropores were synthesized to encapsulate enzyme(horseradish peroxidase,HRP,which is a common marker in ELISA.Then,an ELISA based on nanoMOFs encapsulated HRP was developed and applied to detect the cancer biomarker(i.e.,prostate specific antigen,PSA).In addition,we also developed a facile method to detect sulfide ion(S2-)by using MOFs with peroxidase activity.Free S2-is widely found in natural water and wastewater,and is a very important water pollution index.It strongly interferes with many biological processes and may cause toxicity to many aquatic organisms even at the micromolar concentration.However,detecting S2-usually requires complex instrumentation and specialized operations.The thesis is mainly composed of the following four parts:Chapter one,introduction.Firstly,through introducing the current status of cancer research,it pointed out the detection methods and monitoring challenges of cancer biomarkers,especially the common ELISA method.The principle,category and application of ELISA were introduced.Secondly,the characterization,synthesis methods and applications of MOFs were summarized.The broad applications of MOFs,especially the catalytic applications,were introduced.Thirdly,the methods and carriers for the immobilization of enzymes were introduced,and the applications of immobilized enzymes using MOFs.Finally,the purpose,strategy and content of study were proposed.In chapter two,according to the reported solvothermal method,we had synthesized PCN-333 with ultra-large mesoporous cage,high porosity and stability,and several microns in size.In order to further synthesize PCN-333(size less than 300 nm)suitable for ELISA experiments,we optimized the size of PCN-333 through a bottom-up approach.It included three methods:diluting the system,adding PVP and diluting the system,and microdroplet reaction synthesis.PCN-333(Al)with 270 nm particle size,uniform morphology and good monodispersity could be synthesized by adding PVP40000 and diluting the system.We controlled the nucleation and growth of PCN-333 through microdroplet reaction,the uniform and monodispersive nanoPCN-3 3 3(Fe)could be synthesized.In addition,we encapsulated HRP using synthesized PCN-333,it can not only immobilize HRP stably,but also preserved the immobilized HRP maintain high activity in low concentration and long time.The Vm value and Kcat value of the HRP@PCN-333(Al)with TMB-H2O2 as substrates were 2.20×10-5 mM/s and 2.20×104 min-1,respectively.They were slightly lower than Vm value(2.03x10-4 mM/s)and Kcat value(1.42×105 min-1)of the free HRP with TMB-H2O2 as substrates.The catalytic activity of immobilized HRP was not significantly affected,and the catalytic efficiency was still high.In chapter three,a sandwich ELISA for PSA detection based on HRP@PCN-333 was developed.For the first time,HRP@PCN-333 was used as a tag to combine with secondary antibody to form enzyme-labeled antibody complex,which catalyzed the reaction of TMB substrate solution to realize color signal amplification to detect PSA.Enzyme-labeled antibody complexes maintained a high catalytic efficiency.Its Vm value and Kcat value with TMB-H2O2 as substrates were 4.84×10-5 mM/s and 4.84×104 min-1,respectively.The results of PSA detection by this method shown that the linear range was 15?165 pg/mL,the coefficient of variation(n=5)was less than 9.5%,with good reproducibility,and the detection limit was 6 pg/mL.It showed the feasibility of this method for PSA detection.In addition,the reliability of the method was further illustrated by the results of recovery experiments.The main characteristics of this work included,enzyme markers increased significantly,the stability and utilization of enzyme improved,the preparation process of enzyme-labeled antibody became simpler,and low cost.These were illustrated the potential application of this work in detecting disease markers under clinical conditions.In chapter four,a colorimetric method for the determination of S2-was developed based on PCN-222(Fe).In the presence of H2O2,peroxidase-like PCN-222(Fe)could catalytic oxidation of TMB to OxTMB(oxidized-TMB),while S2-could reduce OxTMB to TMB,and the interaction of S2-with Fe3+in PCN-222(Fe)decreased the catalytic activity of PCN-222(Fe)which inhibited TMB oxidation.The linear range of detection and the limit of detection of S2-were 0.02?0.5 mM and 13.9 M,respectively.In addition,this method had good selectivity for the detection of S2-without interference from other ions.Obviously,combining the characteristics of PCN-222(Fe)and S2-,it could detect S2-simply and sensitively. |
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