A core--Shell-type flourescent nanosphere possessing reactive poly(ethylene glycol) detection of protein in time-resolved fluorometric immunoassay

To increase the sensitivity and to depress the nonspecific binding in biochemical assays, a new core-shell-type fluorescent nanosphere (106.7 nm) covalently conjugated with antibody was prepared. The core-shell-type nanosphere was constructed by dispersion radical polymexization of styrene in the presence of heterotelechelic poly(ethylene glycol) (PEG) macromonomer, which has a polymerizable vinylbenzyl group at one end and a primary amino group at the other chain end and used as well as a surfactant. The resulting nanosphere had PEG tethered chains on the surface, which possesses a primary amino group at the distal end of the PEG chain (NH2 nanosphere). The fluorescent NH2 nanosphere was constructed by incorporating fluorescent europium chelates with 6-diketonate ligands in the core of the NH2 nanosphere by means of a physical entrapment method. Ile primary amino groups on the fluorescent NH2 nanosphere were then converted to maleimide groups using a hetero cross-linker. The resulting naniosphere had maleimide groups on the surface (maleimide nanosphere), onto which proteins having SH group in the molecule could be covalently conjugated quantitatively without any dena_ turation of the proteins under the milder reaction condition. The applicability of the fluorescent nanosphere was tested in a model sandwich immunoassay for a-letoprotein (AFP) determination. Anti-human AF`P Fab'fragment was covalently conjugated onto the maleimide nanosphere (Fab' nanosphere), and it was used for the solid-phase time-resolved fluorometric immunoassay of AFP. The detection limit (mean + 2 SD) was 0.040 pg/mL or 57.1 zmol (57.1 x 10-21 mol, M,,A,~T = 70 000) for AFP. The imprecision (concentration CV) over the whole assay rangewas 1.1% (100pg/nflL) - 17.1% (0.1 pg/mL), even though with this conjugation of antibody to the nanosphere, the nonspecific binding was practically negJJigible (0.0008%) and even when -1.9 x 109 particles of the Fab' nanosphere were applied to the microtitration well. [PERIODICAL ABSTRACT]