Preparation Of Multi-color Monodisperse Fluoresent Microsphere

The fluorescent microspheres were synthesized by Drying Method in Liquid and Polymerization Method, respectively. Fluorescein isothiocyanate (FITC) loaded microspheres were prepared by improved Drying Method in Liquid (oil-in-water, O/W) using Polystyrene (PS) as carrier, F-68 as emulsifier. The fluorescent microspheres were characterized for yield, particle size, coefficient of variation (Cv) and fluorescent intensity. The influence factors, including preparation method, oil and water phase, the concentration of FITC, were discussed. With a mathematic model, the efficiency of washing process was analyzed. The higher efficiency of washing was gained when using 30mL liquid in every washing process.The Polymerization Method divided into monodisperse polymerization and seed polymerization were prepared as follows: Firstly, the highly uniformed polystyrene (PS) seed particles were produced by monodispersion polymerization using an initiator, 2,2-azobis- (isobutyronitrile) (AIBN) in alcohol media. The particle size of seeds was 3.12μm with the Cv less than 2%. Secondly, Monodisperse polystyrene seed particles were redispersed in sodium dodecyl sulfate (0.25wt%, SLS) aqueous solution by sonication, swollen with Phthalic acid dibutyl ester (DBP) and chloroform (totally 0.05g) pre-emulsified by ultrasonic for 10h at 20℃. Then, the mixture of BPO (as initiator, 1wt% relative to styrene) and styrene monomer (10g) was poured into the reactor. The swelling was continued for another 6 h at 20℃. After deaerated with nitrogen gas, the polymerization was carried out at 60℃for 10 h. The size of particles produced by seed polymerization was 9.9μm with the Cv less than 4%. Finally, The PS beads were swellen by chloroform and 1-propanol at room temperature for 24h with light shading equipment, meanwhile, labeled by fluoresceins (rhodamine and acridine orange). The labeled beads were washed with alcohol for three times to remove the remainder. The influence factors, such as polymerization method, polymerization temperature, the concentration of styrene, fluoresceins and Poly(vinyl pyrrolidone) (PVPK-30), were discussed.Using dynamic balance model, the process of the forming of primary particle and mechanism of monodisperse polymerization was elucidated. Choosing the best groups of paralleled experiment data, using genetic algorithms (GA) method, the optimal swellen parameters, which were 10 (V/V) percents of chloroform, 30mmol fluorescein, 33 percents of AO and swellen for 24h were obtained in sixth generation. Fourier Transform Infrared Spectroscopy (FTIR) analysis indicated the fluoresceins were absorbed to the inner of the PS particles, and the surface didn't detected any obviously residual fluoresceins. The morphology of fluorescent beads didn't have any visible difference from the unlabeled PS particles'. The products were excitated at 490nm to various colors, such as red, green, blue et al.., with the range of emission wavelength between 510 and 500 nm, qualified as absolute count reagent for flow cytometry analysis.