Poly(styrene-acrylamide-acrylic acid) copolymer fluorescent microspheres with improved hydrophilicity: Preparation and influence on protein immobilization

Poly(styrene-acrylamide-acrylic acid) copolymer fluorescent microspheres (PSAAFMs) with improved surface hydrophilicity were synthesized through an improved soap-free emulsion copolymerization method, in which the proportion of acrylamide on the surface of the microspheres was increased. Azidocarbonyl groups, which can be rapidly coupled with proteins under mild conditions, were introduced onto the PSAAFMs using an azido reaction. The PSAAFMs were characterized using a fluorescence microscope, an ultraviolet/visible spectrometer, a Fourier transforms infrared spectrometer, a transmission electron microscope (TEM), a size analyzer, and a fluorescence spectrophotometer. Furthermore, covalent linking through the azidocarbonyl groups and physical nonspecific attachments of bovine serum albumin (BSA), trypsin, and human chorionic gonadotropin (HCG) onto the surface of the microspheres were also determined to evaluate the influence of improved surface hydrophilicity on nonspecific protein adsorption. Results from the TEM and size analyzer showed that the PSAAFMs maintained spherical shapes with an average diameter of 2.5 ± 0.22 μm. Fluorescence measurement indicated that the maximum emission wavelength underwent a slight blue shift from 514 to 512 nm. Environmental factors, such as pH value, imposed certain effects on fluorescence intensities. The linear relationship between fluorescence intensity and microspheres' concentration, which ranged from 1 × 10^-3 to 10 × 10^-3 g L^-1, suggest their quantitative application. The significant decrease in the physical nonspecific adsorption of BSA, trypsin, and HCG in comparison with the microspheres without improved hydrophilicity suggest the increased amount of acrylamide on the surface of the microspheres. The protein covalent immobilization experiments revealed significant increases in BSA and HCG immobilization in comparison with the nonspecific physical attachment. The combination of high hydrophilicity and electrostatic repulsion could severely inhibit nonspecific protein attachment onto the surface of the microspheres.