Determination of Human Alpha-Fetoprotein (AFP) by Solid Substrate Room Temperature Phosphorescence Enzyme-Linked Immune Response Using Luminescent Nanoparticles

Abstract

Luminescent nanoparticles of silicon dioxide (SiO2) containing fluorescein isothiocyanate (FITC) with a particle size of 20 nm were synthesized using the Sol–Gel method (abbreviated FITC–SiO2). FITC–SiO2 nanoparticles whose surfaces are modified (FITC–SiO2–S–CH2COOH) can emit strong and stable solid substrate room temperature phosphorescence (SS-RTP) on acetyl cellulose membranes. When the original color-producing agent (R) in the enzyme-linked immunosorbent assay (ELISA) kits for determination of alpha-fetoprotein (AFP) was substituted with (FITC–SiO2–S–CH2COOH), the system maintained good FITC–SiO2 phosphorescence properties. Furthermore, the phosphorescence intensity enhanced markedly after the ELISA reaction. The relationship between the phosphorescence intensity and the content of AFP obeyed Beer’s law. Based on the facts stated above, a new method for the determination of human AFP by SS-RTP-ELISA (using the luminescent nanoparticle as marker) was established. The linear range of this method is 0.040–16.0 pg of human AFP per spot (sample volume: 0.40 µL spot−1, corresponding concentration: 0.10–40.0 ng mL−1). The regression equation of the working curve is ΔIp = −6.289+18.075 mAFP (pg spot−1) (r = 0.9960, n = 6). The detection limit (LD) of this method calculated by 3 Sb/k is 6.7 fg spot−1 (corresponding concentration: 17 ng L−1). Compared to the ELISA method using a traditional color-producing agent, the new method exhibited a 34.8 times higher sensitivity and a wider linear range. The method has been successfully applied to the determination of human AFP in serum.