A new time-resolved fluorometric microarray detection system using core-shell-type fluorescent nanosphere and its application to allergen microarray.

We have developed a new time-resolved fluorometric (TRF) microarray detection system consisting of fluorescent NH2 nanosphere, TRF microarray detector and gamma-irradiated polystyrene chip. Using the TRF microarray detector, we detected 500 particles of the fluorescent nanosphere in one channel. Cross-talk fluorescence from the adjacent channels was little observed in the TRF microarray detector (<0.0004 %). The TRF microarray detection system was further applied for serum allergen-specific immunoglobulin E (IgE) multi-analyses. As a labeled tag antibody, an anti-human IgE Fab' fragment-conjugated fluorescent nanosphere (Fab' nanosphere) was prepared as described previously. As a chip surface appropriate for allergen immobilization, the polystyrene chip surface was modified by gamma irradiation. The immunoassay reactivity using the gamma-irradiated polystyrene chip was approximately 2.5-times improved compared with that of the non-treated polystyrene chip. Non-specific adsorption of the Fab' nanosphere onto the gamma-irradiated polystyrene chip surface was very low level (<0.0009 %). In only 20 mul of serum, six allergen-specific IgEs could be simultaneously determined in one reaction well in fewer than 90 min. Good correlation curves were obtained between the microarray immunoassay and the CAP RAST fluoro-enzyme immunoassay (CAP/RAST FEIA) method (r > 0.961). Reproducibility (CVs) of the microarray immunoassay was 8.6 % to 19.0 % (n = 5).