Synthesis Of Dyes Used For Fluorescent Microspheres And The Preparation Of Fluorescent Microspheres

Flow cytometry (FCM) is a new, fast-growing biomedical analysis technology. In recent years, with the combination of flow cytometry and fluorescent microsphere, a newly developing technology known as flow cytometry based fluorescent microsphere emerged. This technology can be combined with a variety of immunoassay methods to achieve the detection and screening of a variety of antigens or virus at the same time, which greatly improves the analysis efficiency.Preparation of fluorescent encoded microspheres is the core of flow cytometry based fluorescent microsphere technology. The fluorescent dyes used to label microsphere are required with large Stokes Shift. Early studies of our research group have found that heptamethine cyanine with amino substitution at the central position has large Stokes Shift. Based on the previous work, a 2,3,3-trimethyl-3H-indole matrix dye with the introduction of benzyl group at N atom of both ends to improve the photostability was synthesized. Five non-hydrophilic amino-substituted heptamethine cyanines were obtained by nucleophilic substitution with amine reagents. The Stokes Shift of the four aliphatic amino-substituted dyes is greater than 100 nm, and fluorescence quantum yield is about 0.1; the aromatic amino-substituted dye has better photostability, but the fluorescence quantum yield is low.Considering the poor photostability of heptamethine cyanine, two stable fluorescent dyes used for the preparation of fluorescent microspheres by emulsion polymerization were synthesis. The one used in encapsulating method has an easy way for preparation, and fluorescence quantum yield can reach 0.6; the one in copolymerization method has a fluorescence quantum yield of about 0.1 owing to the photoinduced electron transfer (PET) phenomenon. pH titration showed that with the increase of acidity, fluorescence markedly increased, and the Stokes Shift of the dye is up to 100 nm. Transmission electron microscopy (TEM) showed that the microspheres obtained by the two methods had uniform size distribution with a diameter of about 150 nm; the microspheres prepared by encapsulating method have core/shell structure, and those by copolymer method have solid structure.In the fourth part of the thesis, reactive dyes were introduced to label bovine serum albumin (BSA) before native polyacrylamide gel electrophoresis. The results show that blue KN-R is the best dye in protein staining and the optimum reaction conditions are pH 10.0,60 ℃,50 min. The excellent reproducibility and stoichiometry within the test scope are obtained and the detection limit to BSA is 200 ng. The method is sensitive, simple, direct and convenient to be observed by naked eyes in real-time style and has good application prospects for the use as protein label agent.