Magnetic Nanoparticles-based Bioseparation Methods And Their Application In Fluorescence Immunoassay

Silica-coated iron oxide nanoparticles?Fe₃O₄@SiO₂ MNPs?,which are magnetic particles possessing superparamagnetism and excellent chemical stability,can be easily modified with amino groups on their surface by amino-silane treatment,followed by effective immobilization of many kinds of chemical and biological molecules or materials via reactions of the immobilized amino groups and other chemical groups of the molecules to be immobilized,to build a magnetic probe.After the chemical or biological recognition of targets by the magnetic probe,an external magnet can be employed to provide the magnetic field for the rapid separation of the composite containing the magnetic probe and the bound targets.Due to the above mentioned features,magnetic bioseparation methods based on Fe₃O₄@SiO₂ MNPs have been widely used in bioanalysis fields.In this work,two kinds of magnetic bioseparation methods based on Fe₃O₄@SiO₂MNPs were developed and applied to build magnetic fluorescence probes for competitive immunoassay of target proteins in complex matrix.?1?Core-shell structured Fe₃O₄@SiO₂ MNPs were synthesized by chemical coprecipitation,followed by functionalization with amino groups and immobilization of antibodies.The synthesized Fe₃O₄@SiO₂-antibody nanoparticles were employed as the probe for the competitive immune recognition of the target antigens in samples and the antigens labeled with fluorescein isothiocyanate?FITC?.After the magnetic separation of probes binding with these two types of antigens,fluorescence of the free FITC-labeled antigens was measured for the quantification of the target antigens,since the ratio of the FITC-labeled antigens in supernatant before and after the competitive immune recognition depends on the amount of the target antigens in sample,due to the competitive nature of the antibody binding for these two types of antigens.Under the optimal conditions,a linear relationship between the change of fluorescence intensity and the concentration of pig IgG was obtained in a range from 0.75 to 23.50?g L^-1,with a detection limit?LOD?of 0.031?g L^-1.?2?Nitrogen-doped graphene quantum dots?N-GQDs?were employed for the modification of the synthesized Fe₃O₄@SiO₂-antibody nanoparticles,to build a new type of magnetic fluorescence probe for the competitive immune recognition of the target antigens in samples and the spiked FITC-labeled antigens.After the recognition reaction,the probes binding with these two types of antigens were rapidly separated from matrix by using an external magnet,followed by being redispersed in buffer and measurement of the fluorescence resonance energy transfer?FRET?between the N-GQDs and FITC immobilized on the probes.The intensity ratios of fluorescence emitted by FITC?528 nm?and N-GQDs?427 nm?were calculated for the following calculation of the amount of the target antigens in sample based on the principle of the competitive immunoassay.Relationship between the fluorescence intensity ratios and the human IgG concentrations obtained by the propose method is linear in the concentration range of 0.005-45?g L^-1?gL^-1,with a LOD of 0.0013?g L^-1.These proposed methods involving magnetic bioseparation and competitive fluorescence immunoassay are suitable for the specific detection of trace target proteins in complex matrix.Method?2?especially demonstrates an outstanding sensitivity performance due to the applying of FRET and ratiometric fluorescence,which indicates its great potential for the determination of trace targets in bioanalysis applications.