Layer-by-layer construction of novel biofunctional fluorescent microparticles for immunoassay applications

A novel class of biofunctional fluorescent microparticles for application i n immunoassays was constructed by using the layer-by-layer self-assembly me thod to deposit multiple layers of fluorescently labeled polyelectrolytes o nto colloidal particles, followed by deposition of a protein (immunoglobuli n G, IgG) layer. Microelectrophoresis experiments revealed alternating nega tive and positive zeta -potentials with deposition of each successive polye lectrolyte layer, indicating that the alternate electrostatic adsorption of polyelectrolytes of opposite charge was successfully achieved. Transmissio n electron microscopy images showed a change of the particle surface textur e after polyelectrolyte multilayer deposition. Fluorescence microscopy imag e (FMI) analysis provided direct measurement of the fluorescence intensity of single microparticles. The observed systematic increase of the fluoresce nce intensity of individual microparticles with increasing polyelectrolyte layer number from FMI analysis further demonstrated the controlled regular adsorption of polyelectrolyte layers onto the polystyrene (PS) particles. P rotein immobilization onto the polyelectrolyte multilayer-coated particles was verified by the different surface properties of the microparticles with respect to surface charge under pH conditions above and below the isoelect ric point of the proteins. The assembly of IgG and fluorescein isothiocyana te-labeled IgG onto polyelectrolyte multilayer-coated PS microparticles and their potential use was ultimately confirmed by a solid phase immunotest. (C) 2001 Academic Press.