Integrated Detection Antibody With Metal-organic Framework For Immunoassay

Enzyme-linked immunoassay has become the gold standard in immunoassays due to its high detection sensitivity,good stability,and simple operation.However,as people’s needs for early diagnosis become higher and higher,the demand for sensitivity has also increased.As a result,the current commercial enzyme-linked immunosensors can no longer meet the demand.In addition,there are also problems in the preparation of enzyme-labeled antibodies.Traditionally,the connection between antibodies and enzymes is achieved through covalent bonding.Although such a combination is tight,the steps are cumbersome and lengthy,making the enzyme and antibody inevitably lose part of the activity.In order to solve the problems of low sensitivity of enzyme-linked immunoassay and complex preparation process of enzyme-labeled antibodies,in this paper,metal-organic frameworks(MOFs)with high porosity,large specific surface area and adjustable performance will be used to self-assemble enzymes and antibodies.This simple synthesis method greatly retains the catalytic performance of the enzyme to the substrate and the recognition ability of the antibody to the antigen,and the catalytic activity of the enzyme-labeled antibody is higher due to the increased ratio of the enzyme to the antibody.Using this complex to carry out enzyme-linked immunoreaction for qualitative and quantitative detection of prostate antigen and achieved good results.main tasks as follows:(1)We embed urease in MOFs and obtained a catalytically active enzyme-MOFs complex.Even after treatment with organic solvents and high temperature,it still maintains good activity.After verifying that the complex has good catalytic stability,goat anti-rabbit immunoglobulin was immobilized on its surface by Schiff base reaction,and finally a recognition-catalytic dual-functional immune complex was obtained and applied to fluorescence.In the immunoassay,experiments prove that the immunosensor has good sensing performance.Through the experimental results,it can be obtained that the embedding or attachment of MOFs to biological macromolecules will not reduce their catalytic or recognition functions but can protect the proteins embedded in them.In addition,in the detection process of the actual sample,we found that it can accurately detect the content of the analyte in the presence of a higher concentration of interfering substances.Accordingly,the complex is an excellent immunoassay complex.(2)In this work,we embed glucose oxidase and horseradish peroxidase in MOFs at the same time.Since MOFs provide a restricted environment for the catalytic reaction of enzymes,the reaction rate of enzymes has been greatly improved.Subsequently,the aldehyde group of the organic ligand Imidazole diformaldehyde for the synthesis of MOFs and the amino group of goat anti-rabbit immunoglobulin were used to perform Schiff base reaction to fix the globulin on the surface of MOFs,thereby obtaining a catalytic complex with immune recognition function.After that,we use the complex in colorimetric immunity and perform qualitative and quantitative detection of prostatespecific antigen.The detection result of the immunosensor is highly consistent with the detection result of a commercial enzyme-linked immunosensor,and the detection limit and detection range are better than those of commercial ELISA,so the sensor has good application prospects.(3)We use positively charged protamine to adsorb luminol and fluorescein sodium,which are both negatively charged,and embed the obtained complex in MOFs.This realizes the uniform distribution of luminol and sodium fluorescein in MOFs and controls the distance between molecules within 10 nm.After chemiluminescence energy transfer of the obtained complex,it is found that the energy transfer efficiency is significantly high.In the control group that did not use protamine.Subsequently,goat anti-rabbit immunoglobulin was modified on the surface of MOFs,and this immune complex was used to detect prostate-specific antigen.The experimental results showed that the immunosensor has good detection sensitivity and anti-interference ability.