Nanoencapsulated microcrystalline particles for superamplified biochemical assays.

We report on the preparation and utilization of a novel class of particulate labels based on nanoencapsulated organic microcrystals with the potential to create highly amplified biochemical assays. Labels were constructed by encapsulating microcrystalline fluorescein diacetate (FDA; average size of 500 nm) within ultrathin polyelectrolyte layers of poly(allylamine hydrochloride) and poly(sodium 4-styrenesulfonate) via the layer-by-layer technique. Subsequently, the polyelectrolyte coating was used as an "interface" for the attachment of anti-mouse antibodies through adsorption. A high molar ratio of fluorescent molecules present in the microcrystal core to biomolecules on the particle surface was achieved. The applicability of the microcrystal-based label system was demonstrated in a model sandwich immunoassay for mouse immunoglobulin G detection. Following the immunoreaction, the FDA core was dissolved by exposure to organic solvent, leading to the release of the FDA molecules into the surrounding medium. Amplification rates of 70-2000-fold (expressed as an increase in assay sensitivity) of the microcrystal label-based assay compared with the corresponding immunoassay performed with direct fluorescently labeled antibodies are reported. Our approach provides a general and facile means to prepare a novel class of biochemical assay labeling systems. The technology has the potential to compete with enzyme-based labels as it does not require long incubation times, thus speeding up bioaffinity tests.

Layer-by-Layer Construction of Novel Biofunctional Fluorescent Microparticles for Immunoassay Applications.

A novel class of biofunctional fluorescent microparticles for application in immunoassays was constructed by using the layer-by-layer self-assembly method to deposit multiple layers of fluorescently labeled polyelectrolytes onto colloidal particles, followed by deposition of a protein (immunoglobulin G, IgG) layer. Microelectrophoresis experiments revealed alternating negative and positive zeta-potentials with deposition of each successive polyelectrolyte layer, indicating that the alternate electrostatic adsorption of polyelectrolytes of opposite charge was successfully achieved. Transmission electron microscopy images showed a change of the particle surface texture after polyelectrolyte multilayer deposition. Fluorescence microscopy image (FMI) analysis provided direct measurement of the fluorescence intensity of single microparticles. The observed systematic increase of the fluorescence 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. Protein 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 isoelectric point of the proteins. The assembly of IgG and fluorescein isothiocyanate-labeled IgG onto polyelectrolyte multilayer-coated PS microparticles and their potential use was ultimately confirmed by a solid phase immunotest. Copyright 2001 Academic Press.

Dispersive X-ray Spectroscopy Analysis of Porous beta-TCP Bioceramics after Implantation into Femur.

In this paper, energy dispersive X-ray spectroscopy and element ratio of implanted bioceramic, interface and rabbit femur when the bioceramic implanted into femur were measured by two different scan electron microscopy (SEM) EDXA modes. The changes of element ratio and components of materials and interface implanted into femur were compared. Thorough studies were made on the transformation of TCP from lifeless materials into living bones after implantation, by using SEM-EDXA, Raman spectra and infrared spectra. These results provided rich evidence for understanding biodegration and bone bonding mechanism of calcium phosphate bioceramics.