Solid-substrate room-temperature phosphorescence immunoassay based on an antibody labeled with nanoparticles containing dibromofluorescein luminescent molecules and analytical application

Luminescent 20-nm silicon dioxide nanoparticles containing dibromofluorescein (D; particles denoted D-SiO2) were synthesized by the sol-gel method. In the presence of Pb(Ac)(2) as a heavy atom perturber, the particle can emit the intense and stable room temperature phosphorescence(RTP) signal on a polyamide membrane at the wavelength of lambda ex(max)/lambda em(max) =460/623 nm for D. Our research indicates that the specific immune reaction between goat-anti-human IgG antibody labeled with D-SiO2 and human IgG can be carried out quantitatively on a polyamide membrane, and the phosphorescence intensity was evidently enhanced after the immunoreaction. Thus a new solid substrate room temperature phosphorescence immunoassay (SS-RTP-IA) for determination of human IgG was established. The linear range of this method is 0.0624-20.0 pg spot(-1) of human IgG (corresponding to a concentration range of 0.156-50.0 ng ml(-1), sample volume: 0.40 mu l spot(-1)). The regression equation of the working curve is Delta Ip=94.39 + 17.00 m IgG (pg spot(-1)) (460/623 nm, r = 0.9998). Detection limit calculated as 3 Sb/k is 0.015 pg spot(-1). After elevenfold replicate measurement, RSD are 3.2% and 2.4% for samples containing 0.156 and 50.0 ng 1(-1) IgG, respectively. This method is sensitive, accurate, and of high precision. And it has been applied to the determination of IgG in human serum with satisfactory results. Meanwhile, the mechanism of SS-RTP-IA based on an antibody labeled with nanoparticles containing dibromofluorescein luminescent molecules was discussed. (c) 2005 Elsevier B.V. All rights reserved.