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

Abstract

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 λ ^max_ex /λ ^max_em =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 μL spot⁻¹). The regression equations of the working curves are ΔI_p=71.27+7.208m_IgG (pg spot⁻¹) (r=0.9996). Detection limits calculated as 3S_b/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.