Preparation Of Fluorescent Microspheres With Functional Groups On The Surface For Bioconjugation Via Layer-by-Layer Technique

Fluorescent microspheres are widely used in the biomedical and clinical fields, while they are very expensive due to the liminted production. Developing simple and effective preparative methods for the fluorescent microspheres is of practical significance. Poly-(p-phenylenevinylene)(PPV) has many advantages such as facile preparation, good photostability and processability. Moreover, its precursor with positive charge can be easily coated onto the surface of negatively charged microspheres. In this paper, we prepared fluorescence microspheres via the layer by layer self-assembly technology, with PPV as fluorescence species. The fluorescence properties and the stability of them were studied. This work including the following two parts:(1)Preparation of crosslinked-multilayer coated fluorescent microspheres with functional groups on the surface for bioconjugationLayer-by-layer technique was employed to introduce poly(phenylenevinylene) precursor(pre-PPV), diazoresin(DAR), poly(sodium-p-styrenesulfonate)(PSS) and poly(acrylic acid)(PAA) onto the substrate polystyrene-divinylbenzene microspheres with sulfonic groups on the surface(SPSDVB). The conversion of PPV precursor into fluorescent PPV as well as the crosslinking reaction between DAR and polyanion, were accomplished simultaneously in the following thermal treatment. After optimizing the DAR concentration, the selection of polyelectrolytes and the coating sequence, the crosslinked multilayer coated PPV microspheres, SPSDVB-(PPV/PSS/DAR/PAA) spheres were prepared. These spheres were found to have uniform size with a clear core-shell structure and display strong fluorescence, based on the characterization by flow cytometry, microscopy and photophysics. They were found to be stable and highly resistant to comment solvents and even “dissociation agent”, as well as possess good thermal stability and photostability. The feasibility of conjugating biomolecules on the surface of spheres was also demonstrated.(2)Preparation of multilayer fluorescent microspheres with polymer micelles as the spacer-layerWe used Atom-transfer radical-polymerization(ATRP) method to synthesize the block copolymer PAA46-b-PS40. Then we obtained the PAA46-b-PS40 micelles suspension via self assembly in a selective solvent. The final resulting micelles are highly uniform in size(with a diameter of 80 nm). Layer-by-layer technique was employed to introduce pre-PPV and PAA46-b-PS40 micelles onto the substrate polystyrene-divinylbenzene microspheres with sulfonic groups on the surface(SPSDVB). After optimizing the concentration and the p H of micelles suspension, the SPSDVB-(PPV/PAA-b-PS)n spheres were prepared. These spheres were found to have uniform size with a clear core-shell structure and display strong fluorescence. These spheres possess good thermal stability and photostability.