The Study Of A Novel Method In Bioanalysis Based On Magnetic And Fluorescent Nanoparticles

This paper has studied the preparation and property of two kinds of fluorescentnanocomposites contained CdTe or CdTe/CdS quantum dots, the synthesis andfunctionalization of magnetic nanoparticles and their applications in the detection ofhepatitis B e antigen （HBeAg） and surface antigen （HBsAg） simultaneously.The silica nanoparticles with the average diameter of60nm were synthesized byStober method, which had good water-solubility and monodispersity. Two kinds offluorescent nanocomposites were got by coating silica nanoparticles with cationicpolyelectrolyte polydiallyldimethylammonium chloride （PDAC）, CdTe or CdTe/CdSquantum dots, PDAC and anionic polyelectrolyte polyacrylic acid （PAA） vialayer-by-layer self-assembly. The adsorption process of quantum dots was optimized.The morphology and quality of the fluorescent nanocomposites were characterized byfluorescence spectrophotometer, transmission electron microscopy （TEM）, Zetapotential analyzer. It is shown that the fluorescent nanocomposites had strongfluorescence, good hydrophilicity and fluorescence stability, low leakage, and weakoptical bleaching. Besides, they had high fluorescence stability against quench causedby acid, alkali, and oxidation. Furthermore, the fluorescent nanocomposites were able toconjugate with biological molecules with the help of carboxyl group, which could beused as probes for detecting dual-antigen at the same time.The Fe₃O₄nanoparticles with superparamagnetism were synthesized bycoprecipitation method. The carboxyl-modification on the surface of the nanoparticleswere conducted by layer-by-layer self-assembly and surface polymerization respectively.The morphology, composition and quality of the obtained nanoparticles werecharacterized by infrared spectrophotometer, X-ray polycrystalline diffraction, TEM,Zeta potential and size distribution analyzer. It is shown that both of two methods ofcarboxyl-modification had no significant effect on the crystallinity of Fe₃O₄, however,improved obviously the dispersibility of the magnetic nanoparticles. Meanwhile, the nanoparticles could be used as magnetic markers in the immunoassay by theconjugation of carboxyl and biological molecules.The conditions of conjugation of two kinds of the fluorescent nanocomposites andthe carboxyl-modified magnetic nanoparticles with their corresponding antibodies,which was activated by the carbodiimide cross-link technique, were investigated.Besides, the conjugation abilities of two kinds of carboxyl-modified magneticnanoparticles with antibodies were compared. The goat anti-HBeAg and anti-HBsAgantibodies labelled by CdTe and CdTe/CdS fluorescent nanocomposites respectively,were used as detection probes. The murine anti-HBeAg and anti-HBsAg antibodieslabelled by magnetic nanoparticles were used as capture probes. Based on thedual-antibody sandwich-type immunoassay, the residual fluorometric detection methodfor HBeAg and HBsAg was established. The linear range of HBeAg was20.0-100.0ng/mL （R2=0.9958）, and HBsAg was20.0-100.0ng/mL （R2=0.9972）. Both of thedetection systems had good accuracy and precision. The results of the cross-reactivity ofantibodies and the selectivity of systems indicated that the simultaneous detection ofHBeAg and HBsAg was feasible. Analytical results of simultaneous determination ofdual-antigen in spiked samples showed that this method was simple, fast, sensitive andcould detect HBeAg and HBsAg simultaneously, which might be applied in detecting ofviral hepatitis B disease in clinical medicine.