Preparation, Characterization Of Functionalized Polystyrene Microsphere And Its Application In Biological Detection

Microsphere-based array, also named as liquid chip or suspension array, is a powerful platform for multiplexed analysis of complex samples. Nucleic acid hybridization, immuno reaction, enzyme reaction can all be carried out on this platform. The suspension array is created by immobilizing capture reagents on the surfaces of encoded microspheres in conjunction with flow cytometric analysis. The array elements, the microspheres, are classified by their distinct optical properties, such as light scatter or fluorescence from an internal dye. When analyzed by the flow cytometer, the microspheres are passed through the laser detection volume one by one. The fluorescence produced from the internal dye is used to decode the microspheres for quanlitative analysis, meanwhile the fluorescence signal from the analyte is used for quantitative analysis.Carboxyl functionalized polystyrene particle is an important carrier in the Microsphere-based array. The external carboxyl group can be activated to couple with probes such as proteins or nucleic acids. The microspheres coupled with probe molecules capture the analytes which then bind with fluorescently labelled reported molecules. Through the detection of the microsphere fluorescence intensity, the analyte concentration can be determined.The main work in this thesis is the synthesis of mono-dispersed and size-controllable polystyrene particles rich in surface carboxyl groups. We also explored the synthesis of cross-linked polystyrene microspheres embeding fluorescent dye. This thesis is composed of four chapters. Chapter one is the overview. Chapter two decribes the synthesis of size-controllable polystyrene microspheres. Chapter three discusses the preparation of carboxyl functionalized polystyrene particles and their application in the detection of alpha-foetal protein (APP). Chapter four decribes the synthesis of cross-linked polystyrene microspheres and their fluorescent dyeing.In Chapter one, the history, development and advantages of biochip are introduced with emphasis on the principle and applications of liquid chip. There are many methods to synthesize carboxyl functioanlzied polystyrene microspheres, the main carrier of liquid chip. The polymerization mechanism, advantages and limitations of several important synthesis methods were reviewed. For the disperse polymerization approach, factors affecting microsphere diameter and monodispersity were discussed. In the end of the introduction section, different approaches for surface carboxyl functionalization of polystyrene particles were discussed, along with the their applications in biological detectionChapter two describes the synthesis of size-controllable polystyrene particles. Polystyrene microspheres with different sizes are used in different fields. The microspheres employed in the liquid chip are in the micron size. So, we aimed to synthesize micro-sized polystyrene particles. In this part of work, through adjusting the wt% of the monomer, the wt% of the MAA (to St), and the concentration of the stabilizer PVP, we were able to successfully synthesize polystyrene microspheres with controllable size in the range of 2.0μm to 7.0μm with good monodispersity.In chapter three, the carboxyl functionalized polystyrene microspheres are prepared. Disperse polymerization, stepwise disperse polymerization, seeded polymerization, two-step polymerization and post-modification were all explored to synthesize the carboxyl functionalized polystyrene microspheres. The first four methods were all limited in synthesizing mono-dispersed polystyrene microspheres with high surface carboxyl density. It was demonstrated that during the post-modification process, the uniformity or diameter of the microsphere was not affected while enhancing the carboxyl groups density on the surface. Therefore, post-modification was recommended as the best approach for obtaining carboxyl functionalized microsphreres.In Chapter four, exploration of preparing cross-linked polystyrene microspheres was carried out. Rhodamine B was embedded in the cross-linked microspheres successfully.The main contribution of this thesis is the synthesis of the size controllable, monodispersed polystyrene microspheres. After post-modification for surface carboxyl functionalization, comparable coupling capability of monoclonal antibody onto the surface was achieved with that of the commercial products sold in US. When tested in AFP detection of biological samples, low detection limit was obtained indicating the potential application of our carboxyl functionalized microspheres in microsphere-based array technology.