RESUMEN
In the presence of ion perturber LiAc, 4-generation polyamidoamine dendrimers (4G-D) could emit strong and stable room temperature phosphorescence (RTP) signal at lambda(max)(ex)/lambda(max)(em) = 511.8/675.3 nm on nitrocellulose membrane (NCM), and Triton X-100 could sharply enhance the RTP signal of 4G-D. Triton X-100-4G-D was used to label concanavalin agglutinin (Con A) to get the labeling product Triton X-100-4G-D-Con A. Quantitative specific affinity adsorption (AA) reaction between Triton X-100-4G-D-Con A and alpha-fetoprotein variant (AFP-V) could be carried out on the surface of NCM, whose product Triton X-100-4G-D-Con A-AFP-V could emit strong and stable RTP and its deltaI(p) was proportional to the content of AFP-V. According to the facts above, a new affinity adsorption solid substrate-room temperature phosphorimetry (AA-SS-RTP) for the determination of trace AFP-V by Con A labeled with Triton X-100-4G-D was established. Detection limits of this method were 0.23 fg spot(-1) (direct method, corresponding concentration: 5.8x10(-13) g mL(-1)) and 0.13 fg spot(-1) (sandwich method, corresponding concentration: 3.2x10(-13) g mL(-1)). It has been successfully applied to determine the content of AFP-V in human serum and forecast human diseases, for its high sensitivity, long RTP lifetime, good repeatability, high accuracy and little background perturbation with lambda(max)(em) at the long wavelength area. Meanwhile, the mechanism for the determination of trace AFP-V using AA-SS-RTP was also discussed.
Asunto(s)
Pruebas Diagnósticas de Rutina , Mediciones Luminiscentes/métodos , Suero/química , alfa-Fetoproteínas/análisis , Adsorción , Variación Genética , Humanos , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Temperatura , alfa-Fetoproteínas/genéticaRESUMEN
Luminescent 20-nm silicon dioxide nanoparticles containing dibromofluorescein (D; particles denoted D-SiO(2)) 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 lambdaex(max)/lambdaem(max) = 460/623 nm for D. Our research indicates that the specific immune reaction between goat-anti-human IgG antibody labeled with D-SiO(2) 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 microl spot(-1)). The regression equation of the working curve is DeltaIp = 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 l(-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.