Synthesis And Characterization Of Ternary Copolymer Fluorescent Microspheres

Latex agglutination test (LAT) is a simple and rapid method used in clinical diagnosis, which plays an important role in detecting the disease during the early periods. Briefly, antigen or antibody is adsorbed physically or bonded covalently on the surface of microsphere, and then the composite can interact with the corresponding antibody or antigen to appear special coagulation. Therefore, the microspheres with different properties attract considerable attention of many scientific researchers. Owing to using water as the reactive medium, soapless emulsion polymerization brings about hardly environmental pollution. And microsphere has "clear" surface without emulsifier. So soapless emulsion polymerization has become the developing direction of the microsphere research recently. During the polymerization process, microsphere with surfacefunctional groups such as carboxylic, amino, hydroxyl and aldehyde can be synthesized by using different functional monomer. But in comparison with enzyme-linked immunoadsordent assay (ELISA) and radioimmunoassay (RIA), the sensitivity of latex agglutination test is not high enough and it can't be detected quantitatively. In order to overcome these disadvantages mentioned above, we attempted to develop a new method which combined latex agglutination test with fluorescent detection. A novel fluorescent microsphere was synthesized which can both enhance the sensitivity of the test and be used in quantitative detection.In this work, we synthesized Styrene-Acrylamide-Acrylic acid ternary copolymer fluorescent microsphere which adsorbed fluorescein by means of soapless emulsion polymerization. The effects of different factors on properties of microsphere were evaluated by using orthogonal-designing table L₁8 (3⁷). Five factors with three levels were considered in the experiments, which were temperature, agitating rate, the value of pH, ionic strength and reacting time. Morphological and size analysis of microsphere were performed by transmission electron microscopy (TEM) and atom force microscopy (AFM). Fluorescence spectrophotometer was used to determine fluorescent property of microsphere. We also studied the stability of microspheres in existence of acid, base, salt and organic solvent.The synthesized St-AAm-AA ternary copolymer fluorescent microspheres were converted into microspheres with azidocarbonyl on the surface by hydrazinelyzing and azidolyzing. Bovine serum albumin(BSA) was immobilized on the surface of the fluorescent microspheres to prepare the sensitve microspheres. The effects of the three factors including the concentration of BSA, the value of pH and the sensitized time on the amount of protein combined were researched. And we also studied the fluorescent properties of sensitive microspheres.The results of experiments indicate: When temperature is 75℃, the agitating rate is 300 r·min^-1, the value of pH is 7.5, the amount of NaCl is 0.5g and the reacting time is 4h, the synthesized microspheres are stable and monodispersed. The average sizes of the microspheres are(218±3.2)nm.The experimental results reveal that the fluorescent properties of microspheres with fluorescein and the microspheres without fluorescein have conspicuous difference. The fluorescent intensity of the microspheres without fluorescein is so weak that it wouldn't interfere the fluorescent detection of microspheres with fluorescein. And it is ascertained that the maximum exciting wavelength and emitting wavelength are 551.7nm and 557.4nm respectively. When the concentration of the fluorescent microspheres is within 1 × 10-3¹0× 10^-3g·L^-1, the fluorescent intensity has the linear relation with concentration of microspheres. With different pH, the fluorescent peak in the emitting curve of fluorescent microspheres has the immobilized position, but the fluorescent intensity changes obviously. And when pH is 6.0, microspheres has the highest fluorescent intensity. The fluorescent intensity can be decreased in the presence of dissolved oxygen, but the exicting wavelength and emitting wavelength don't...