Rare Earth Complex-Encoded Fluorescent Polystyrene Microspheres: Preparation And Characterization

Suspension array technology has emerged as a powerful tool for high-throughputdetection over the past decade. This technology is based on the use of flow cytometryto detect target bound fluorescence-encoded microspheres at high speed, capable ofdetecting up to100different targeting species in a single run. Because of the speedand throughput, the suspension array technology is believed to play an increasinglyimportant role in fields of life science. The key to success in suspension array is toprepare fluorescence-encoded microspheres with tunable intensities. Currently, theyare prepared by fluorescence dyeing of monodisperse polymer microspheres withorganic dyes or quantum dots. But they both show short fluorescence lifetimes, andrelatively high background fluorescence which is detrimental to detection sensitivity.In contrast, rare earth metal ions exhibit unique optical characteristics, such as longluminescence lifetime, large Stoke’s shift and sharp line-like atomic emission. Thesefeatures can help overcome autofluorescence and light scattering in chemical biology,and improve the detection sensitivity and resolution. The rare earth metal containingfluorescent microspheres can be prepared by embedding or binding methods.However, with these two methods it is difficult to prepare monodisperse microsphereswith well controlled fluorescence intensity.The ultimate goal of this thesis is to develop methods for preparation of rareearth complex containing fluorescence-encoded microspheres with uniform size andprecisely controlled intensity, providing next generation fluorescently encodedparticles for suspension array technolgoy. The specific objectives were as follows:（1）preparation, characterization and functionalization of monodisperse polymermicrospheres;（2） preparation and characterization of rare earth complexes;（3）preparation and characterization of fluorescence microspheres with rare earthcomplexes;（4） preparation and characterization of fluorescence-encodedmicrospheres with rare earth complexes.Firstly, the monodisperse polystyrene microspheres （PS） with different sizes inthe range of50nm-5μm were prepared by emulsion polymerization, soap-freepolymerization or dispersion polymerization. To facilitate conjugation withbimolecules, the microspheres were functionalized with carboxyl or amino groups.Then, the red fluorescent polystyrene microspheres with europium complex （Eu@PS）were prepared by swelling or coating method. The europium complex waseuropium-dibenzoylmethane-phenanthroline （Eu（DBM）₃phen）, which shows good homochromy and stability. The key factors that impact on the morphology andfluorescence intensity of Eu@PS were investigated and the preparation conditionswere optimized. The morphology, size and fluorescent properties of the fluorescentmicrospheres were characterized by environmental scanning electron microscope（ESEM）, transmission electron microscope （TEM）, fluorescence microscope andspectrofluorophotometer. The Eu@PS prepared using the two methods weremonodisperse, and the particles of varying fluorescence intensities were obtained byadjusting the amount of Eu（DBM）₃phen in the reaction mixture. Furthermore, theamino-surface fluorescent microspheres （Eu@PS-NH₂） prepared by the swellingmethod were used for immobilization of trypsin. The fluorescent intensity ofEu@PS-NH₂was unchanged during the conjugation reaction, implying its potential inbiological assays. Finally, a green fluorescent complex, terbium-acetylacetone-phenanthroline （Tb（acac）3phen） was synthesized and incorporated into theeuropium containing microspheres in varying ratios. The fluorescence-encodedmicrospheres （Eu/Tb@PS） of6different intensity ratios were successfully preparedby one-step swelling method and two-step method.In summary, controlled swelling and coating methods can be adapted to prepareeuropium containing fluorescent microspheres with uniform particle size and tunablefluorescence intensity. The europium containing particles can be further impregnatedwith terbium complex to produce fluorescence-encoded microspheres. As expected,the resulting particles show narrow emission bands, tunable fluorescence intensities,and prolonged optical stability, making them an ideal alternative to organic dyecontaining fluorescent microspheres commercially available.