Preparation Of Fluorescence-Encoded Microspheres With Different Types Of Fluorophores

Suspension array technology has wide applications in the biological and medical fields. Comparing to the traditional detection technology, the suspension array offers many distinct characteristics, such as more efficient interaction with the analytes, faster and easier washing/separation process, and less the sample volume, and, in particular, the simultaneous multiplexed detection in a very short time. As the “heart” of this technology, the beads(or microspheres) need to be encoded to ensure the efficient identification of the interaction between analytes and spheres. The fluorescent microspheres are used most due to their unique merits of fast signal acquisition and high sensitivity. The growing demand of the fluorescence-encoded microspheres from the disease diagnosis and immunoassay requires more encoded microspheres with size monodispersity. In additional, the surface modification to incorporate biological species is important for the microsphere applied in biological detection. Thus, developing new strategies for producing more encoded microspheres with size monodispersity and their surface functionalization are of practical significance.In this thesis, we first advanced a new encoding strategy that a series of fluorescent microspheres were obtained based on three different types of fluorophores. Then, a series of color tunable fluorescent microspheres with functional groups on the surface were prepared based on conjugated polymers.(1) Preparation of monodispersed and spectrally encoded microspheres with three different types of fluorophoresWe explored a strategy for preparation of spectrally encoded fluorescent microspheres through introducing rare earth europium ion(Eu3+) and organic dye rhodamine 6G(R6G) onto the porous and monodispersed polymer spheres with size of 10 μm micrometers. Various combinations of the three different types of fluorophores, the polymer spheres as a polymer fluorophore, and the inorganic Eu3+ and organic R6 G fluorophores, afforded large encoding capacity based on the emission spectra. After optimization, 10 wavelength-intensity combinations from the binary encoded spheres and 15 combinations from the ternary encoded spheres were obtained. Such combinations(or fluorescence codes) were obtained with the excitation at the same wavelength. The microscopic study showed that the monodispersity in size and smooth surface morphology of the substrate spheres were retained after introducing fluorophores. In addition, these spheres were found to have good stability against washing and thermal treatment.(2) Preparation of color tunable fluorescent microspheres with functional groups on the surface.Fluorescent microspheres with functional groups were prepared through introducing poly(p-phenylenevinylene) precursor with carboxyl groups(pre-PPVCOO-) onto the substrate amino-modified poly(glycidylmethacrylate)(APGMA) microspheres followed by thermal elimination. Through varying the elimination temperature, a series of fluorescent microspheres were obtained with different fluorescence emissions and different apparent colors. Compared with the fluorescent spheres prepared in our previous work, the fluorescent spheres obtaind in this study displayed larger difference in the emission peak. The decoding of the fluorescence intensity obtained from different receiving channels of the flow cytometry endowed the spheres with a series of fluorescent codes. These spheres were demonstrated to have a porous surface, monodispersity, and a core-shell structure; they were stable and highly resistant to “dissociation agent”, as well as possessed good thermal stability and photostability. The conjugation with biomolecules on the surface of spheres was also demonstrated.