Determination of human IgG by solid-substrate room-temperature phosphorescence immunoassay based on an antibody labeled with nanoparticles containing rhodamine 6G luminescent molecules.

Luminescent 50-nm silicon dioxide nanoparticles containing both types of rhodamine 6G (R; particles denoted R-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 of R on a polyamide membrane, with lambda(ex)max/ lambda(em)max=470/635 nm for R. Our research indicates that the specific immune reaction between goat-anti-human IgG antibody labeled with R-SiO2 and human IgG can be carried out quantitatively on a polyamide membrane, and the phosphorescence intensity was enhanced after the immunoreaction. Thus a new method for solid-substrate room-temperature phosphorescence immunoassay (SS-RTP-IA) for determination of human IgG was established on the basis of antibody labeled with the nanoparticles containing binary luminescent molecules. 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 microL spot(-1)). The regression equations of the working curves are DeltaIp = 71.27+7.208 m(IgG) (pg spot(-1)) (r = 0.9996). Detection limits calculated as 3 Sb/k are 0.022 pg spot(-1). Compared with the same IA using fluorescein isothiocyanate (FITC) as the marker the new method was more sensitive and had a wider linear range. After elevenfold replicate measurement RSD are 4.5 and 3.6% for samples containing 0.156 and 50.0 ng mL(-1) IgG, respectively. This method is sensitive, accurate, and of high precision.