| Due to its ability to quantify multiple proteins, cytokines, or nucleic acid sequences in parallel using a single sample, fluorescence-encoded polymer bead-based suspension arrays (liquid chips) are widely used in biomolecular screening and diagnostic applications. The preparation of high-performance polystyrene microspheres and encoded methods play critical roles in the field of suspension array biochip for rapid and multiplex detection.Firstly, the 2μm polystyrene seeds were synthesized by dispersion polymerization. And carboxylated polystyrene microspheres with the diameter in the range of 5-20μm were prepared via seeded polymerization. The parameters influencing the diameter and morphology of microspheres, including the monomer swelling time, the content of styrene and MAA, were investigated. The reactivity of carboxyl group on the surface of microspheres was confirmed by immunoreactions. Secondly, the highly cross-linked carboxylated poly (styrene-co-ethylene glycol dimethacrylate-co-methacrylic acid) beads (PSEMBs) with proper pore sizes were designed, fabricated, and further employed in the preparation of high-performance QD-encoded microbeads via gradual solvent evaporation method. The PSEMBs and QD-encoded PSEMBs were characterized by scanning electron microscopy (SEM), laser scanning confocal microscopy, and spectrofluorometry. Next, we developed a new method which was called high-temperature swelling method to prapare high-stable functionalized barcodes. The physical and chemical stabilities of barcodes were evaluated. Thirdly, an efficiently modified method combined conventional swelling method with high-temperature swelling method was applied to prepare multifunctional MNPs-QD-encoded polystyrene beads simultaneously with large encoding capacity, high-stable performance and fast separation. The effect of the sequence of MNPs and QDs doped into polymers on the fluorescent intensity and magnetization of bifunctional microspheres and the stability of MNPs-QD-encoded PSEMBs in organic solves and different pH buffers were evaluated. Meanwhile, using the human IgG as a model analyte, QD-encoded PSEMBs as immunoassay was also illustrated for diagnostic applications. Finally, the flow cytometry and microfluidic–chip analysis systems for decoding QD-encoded beads were successfully established respectively.The SEM images of PSEMBs demonstrated the good sphericity and uniform particle size distribution. The FTIR and 1~HNMR results indicated that the MAA monomer was successfully copolymerized to the microspheres. The content of carboxyl group on the microsphere surface was 1.95×105 carboxyl/per bead via conductometric titration. The high reactivity of carboxyl group on the surface of microspheres was well confirmed by immunoreactions. Confocal microscope images illustrated that highly uniform bright fluorescent beads are obtained and the quantum dots (QDs) had filtrated into the entire microspheres with proper pore size achieved by adjusting the content of porogen. Furthermore, QD-encoded PSEMBs had been displayed to be photostable and kept their bright fluorescence for at least 20 days. Immunoassay performance for human IgG detections indicated that carboxyl groups on fluorescence microsphere surface facilitate efficient attachment of biomacromolecule and therefore enable high detection sensitivity (0.01 ng/mL) in sandwich immunoreactions. Compared with the QD-encode beads with unsealed pores, the QDs leaching from the QD-encoded beads prepared by high-temperature swelling method was negligible under the organic solvent cyclohexane which can swell the beads and also easily dissolve the QDs. Their fluorescence intensity appeared tiny change in a wide range of pH buffers, especially in a range of pH 5-10, the intensity kept relative constant. Meanwhile, the QD-encoded beads could be stored in PBS for at least two months which showed excellent longtime storage stability. The small fluctuations of their wavelength in multi-color barcodes opens up the possibility of massive multiplex optical coding for demanding biolabelingapplications and immunoassay performance for AFP detections indicated that carboxyl groups on fluorescence microsphere surface facilitated efficient attachment of biomacromolecule. As for multifunctional MNPs-QD-encoded polystyrene beads, the results demonstrated that encapsulating QDs into pre-prepared MNPs-beads was the best sequence, which kept high brightness and fast separation. The MNPs-QD-encoded microcarriers designed by our proposal method exhibited excellent performance for magnetic manipulation and optical encoding; what is more, they also had strong physical and chemical stabilities. The study on the interaction between MNPs and QDs showed that a significant decreasing of fluorescence signals was observed with iron oxide increasing, the best content was at the range of 5-15μg/mg. Meawhile, it was certificated in bead-based immunoassays that MNPs-QD-PSEMBs fabricated via high temperature method still had excellent binding capacity for biomolecules to fit for fast separation and multiplexed assays. In flow cytometry analysis, the microspheres with various sizes and different fluorescent intentsity could be completely separated into different group, which showed the diameter-encoded method or diameter-to-intensity method could be validly employed respectively in the multiplexing detection systems. Meanwhile, by microfluidic–chip analysis systems, the motion of microspheres could be operated and corresponding fluorescence signal could be real-time monitored. This work was beneficial to fabrication of QD-encoded polymer beads for suspension arrays, and this robust study could be further utilized to the field of biomolecular screening and diagnostic applications. |
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