Preparation of streptavidin-coated magnetic nanoparticles for DNA detection

With the properties of magnetic response and surface functionality, core-shell magnetic polymer microspheres have been investigated extensively due to their potential applications in biomedicine and bioengineering. Because of the extremely strong affinity and high specificity of interactions between streptavidin and biotin, the streptavidin-biotin system has been utilized in biological areas of gene detection, biosensors and enzyme-linked immunosorbent assay (ELISA). Therefore, coupling streptavidin to magnetic nanoparticles (MNPs) will greatly expand applications of MNPs. In this paper, amino-modified magnetic nanogels (AmMNGs) were synthesized in one step by photoinitiation and in-situ photopolymerization of allyamine in Fe3O4 aqueous suspension under UV irradiation, a method free of initiators and additives. Then streptavidin-coated magnetic nanoparticles (SAMNPs) were fabricated by covalently coupling SA to the surface of magnetic nanogels, which had been activated by glutaraldehyde. The binding capacity of SAMNPs was calculated from the mole number of biotin p-nitrophenyl ester (BNPE) bound by a certain quantity of SAMNPs via colorimetric measurement. An optimized parameter of the covalent SA coupling was presented by investigating effects of pH value of the reaction system, SA concentration and reaction time on the binding capacities. Moreover, methoxy-substituted poly (ethylene glycol) amine (MPEG-NH2) with low molecule weight was used as a blocking reagent to terminate the residual activated groups, as it had been reported that poly(ethylene glycol) could significantly suppress nonspecific binding in the separation of biomolecules. Subsequently, the obtained SAMNPs were successfully applied to detect a targeted DNA with a concentration of 10 nM according to a 'sandwich-type' DNA detection strategy. And the control experiment indicated that the non-specific binding during the detection process was undetectable. Because fluorescein emission is much more intense in polyfluorene amplification than direct excitation[4], the SAMNPs are expected to detect targeted DNA with a concentration below 10 nM in conjugated polymers amplification. This work was supported by Shanghai Municipal Commission of Science and Technology under Contract No.0452nm068 as a special project of nanometer science and technology. (authors)