A Core−Shell-Type Fluorescent Nanosphere Possessing Reactive Poly(ethylene glycol) Tethered Chains on the Surface for Zeptomole Detection of Protein in Time-Resolved Fluorometric Immunoassay

Abstract

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 polymerization 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 β-diketonate ligands in the core of the NH₂ nanosphere by means of a physical entrapment method. The primary amino groups on the fluorescent NH₂ nanosphere were then converted to maleimide groups using a hetero cross-linker. The resulting nanosphere had maleimide groups on the surface (maleimide nanosphere), onto which proteins having SH group in the molecule could be covalently conjugated quantitatively without any denaturation of the proteins under the milder reaction condition. The applicability of the fluorescent nanosphere was tested in a model sandwich immunoassay for α-fetoprotein (AFP) determination. Anti-human AFP 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 × 10^-21 mol, M_w,AFP = 70 000) for AFP. The imprecision (concentration CV) over the whole assay range was 1.1% (100 pg/mL) − 17.1% (0.1 pg/mL), even though with this conjugation of antibody to the nanosphere, the nonspecific binding was practically negligible (0.0008%) and even when ∼1.9 × 10⁹ particles of the Fab‘ nanosphere were applied to the microtitration well.