| Magnetic microsphere is a new kind of multifunctional material.Because of its polymer microsphere properties and superparamagnetism,it is widely used in the fields of protein molecular purification,immobilized enzyme,nucleic acid extraction,cell separation and immune analysis,etc.However,in the actual process of sample separation,many unnecessary compounds are adsorbed on the surface of microspheres,affecting the accuracy of separation and extraction.In the process of immune detection,non-specific binding of other biological molecules may be generated,resulting in false positive.What's worse,sample dilution may be required due to low sensitivity,increasing the complexity of detection procedures.Therefore,different kinds of macromolecules and biomolecules were modified on the surface of magnetic microspheres in this paper,and the magnetic beads were evaluated by chemiluminescence immunoassay,providing a reference for the application of magnetic microspheres in biological extraction,immunoassay and other fields.Firstly,PAA with different molecular weights and dextran were selected to modify the surface carboxyl groups of amino beads,and PAA 5000 was selected as the optimal group.On this basis,PEG molecules were modified on the magnetic bead surface.After modification,the antibody binding amount increased,the background value of chemiluminescence detection decreased with its the sensitivity increasing.Then,the conditions of PAA and PEG conjugated magnetic beads antibody were optimized.Nonspecific adsorption test showed that PEG modification could reduce the nonspecific adsorption of magnetic beads.Finally,the stability experiments showed that the macromolecular modified magnetic beads and antibody magnetic beads maintained various property stability for 3 months and 45 days respectively.Therefore,PAA and PEG can effectively improve the dispersion stability and immune detection sensitivity of magnetic beads,providing reference for the polymer modification on magnetic beads.Subsequently,PAA-modified carboxyl magnetic beads were used as the basis for surface affinity cross-linking,respectively using the one-step method and the two-step method.The optimal reaction conditions of the two methods were selected,SA with 95 ?g magnetic beads per mg by one-step method and SA with 23.8 ?g magnetic beads per mg by two-step method.The different structures of SA magnetic beads formed by the two methods were deduced:one-step method result in multilayer adsorption,two-step method result in single-layer adsorption.Then,SA conjugation was performed on pegylated magnetic beads,and it was found that SA magnetic beads can reduce the non-specific adsorption in the two-step method.Finally,SA magnetic bead can maintain good stability and the sensitivity of immune detection within 20 weeks,which can be regarded as a good biomolecular functional magnetic bead.Finally,PAA-modified carboxyl magnetic beads were selected as the control group to compare the effect of streptavidin magnetic beads and protein A magnetic beads on antibody coupling.The antibody coupling rate of SA and protein A was above 80% and 39%respectively,both of which were higher than that of carboxyl beads(33%).After that,three kinds of magnetic beads with the same antibody coupling amount were selected for comparison.In terms of detection sensitivity,SA magnetic beads were superior to protein A magnetic beads and carboxyl magnetic beads,while non-specific adsorption of protein A magnetic beads was more serious than carboxyl magnetic beads and SA magnetic beads.Therefore,SA magnetic bead and protein A magnetic bead show higher coupling rate,antibody activity and immune detection sensitivity than carboxyl magnetic bead,providing A new reference idea for the preparation of antibody magnetic bead. |
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