| Avidin is a glycoprotein found naturally in the egg white. Avidin has been widely used because of the antimicrobial activity and insecticidal properities. With the development of Avidin, specific measurement of Avidin concentration is frequently needed. In this paper, two approaches based on magnetic nanoparticles for detecting Avidin had been reported. The main research results as follows:Fe3O4 nanoparticles were obtained via a facile solvethermal synthetic method, The optimum preparing process is as fellows:FeCl3·6H2O(0.2702 g), polyethyleneimine (0.50 g) and NaAc·3H2O(2.70 g) were added to ethylene glycol(20 mL) to form colloid mixture under stirring at room temperature. The mixture was then sealed in a teflon-lined stainless-steel autoclave of 25 mL capacity. Finally, the autoclave was heated and maintained at 200℃for 12 h, and allowed to cool down to room temperature naturally. The black products were washed several times with absolute ethanol and dried. The nanoparticles were shown to contain amino-group by FT-IR, the concentration of amino-group on the surface of nanoparticles were 14.97 nmol/mg. Magnetic hysteresis loops of Fe3O4 nanoparticles showed that the values of saturation magnetization (Ms) were 64.93 emu/g.Immuno-magnetic nanoparticles were prepared by linking Fe3O4 nanoparticles with antibody. The optimal conditions for linkage were as fellow:nanoparticles were actived with 15% glutaraldehyde, the actived nanoparticles linking antibody in PBS(0.05 M, pH 7.4), the maximum linkage rate was about 50μg/mg when the mass ratio between antibody and nanoparticles (μg:mg) was 300:1.A magnetic nanoparticle-based competitive inhibition assay for avidn was developed. The proper concentration of Biotin-MNPs was 400μg/mL and proper dilutability of HRP-Avidin was 1:2000. The standard cruve for Avidin was in the concentration range of 79.4-5000.0 ng/mL. The limit of detection was spproximately 79.4 ng/mL. The cross-reactivity with ysozyme, BSA, ovalbumin and ovotransferrin were all less than 0.2%, The method had acceptable intraasssay(7.22%)and interassay precision(9.17%).A magnetic nanoparticle-based sandwich assay for avidn was developed. The proper concentration of immuno-magnetic nanoparticles was 600μg/mL and proper dilutability of HRP-Biotin was 1:800. The linear equation was y= 0.2725 x-0.7036, R2= 0.9915. The standard cruve for Avidin was in the concentration range of 31.2-10000.0 ng/mL. The limit of detection was 30 ng/mL. Specificity assays revealed that the assay didn't cross-reactivity with lysozyme, BSA, ovalbumin and ovotransferrin.The coefficient of recovery was between 88.2%-99.4%.The concentration of the three varieties Avidin which were detected by the three different methods were around 42-60μg/mL, agree with the theoretical value. Data were analyzed with Pearson Correlation and Paired-samples t Test. And the paired t test showed that there was insignificance(P>0.05) between fluorescence polarization method and competitive inhibition method, it was also insignificance (P>0.05) between fluorescence polarization method and sandwich method. |
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