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An ultrasensitive immunoassay was developed on the basis of As3+ and Hg2+ labeled SiO2@Au nanoparticles (NPs) probe and hydride generation-atomic fluorescence spectrometry (HG-AFS) for the detection of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9). Firstly, amino SiO2@Au NPs were synthesized for selective absorption of As3+ and Hg2 to form As(Hg)-SiO2@Au NPs signal tags, respectively. Subsequently, the prepared probes were labeled on the secondary antibody (Ab2) of CEA and CA 19-9, respectively. Based on the sandwich immunoassay scheme, corresponding Ab1, antigen and Ab2 modified with probes, were conjugated to form the immunocomplex on the bottom of 96-well plate at room temperature. After the immunocomplex were dissolved in an alkali solution, As3+ and Hg2+ were released into the solution and detected by HG-AFS, the atomic fluorescence intensity of As3+ and Hg2+ were proportional with logarithms of CEA and CA 19-9 concentration, then indirect quantitative analysis of CEA and CA 19-9 was realized. This assay was based on the determination of the concentration of As3+ and Hg2+ by HG-AFS for quantization of the corresponding CEA and CA 19-9 antigen. The assay showed a wide linear range of 0.001–100.0 µg L−1 for CEA and 0.01–80 U mL−1 for CA 19-9, with the detection limit of 0.5 ng L−1 and 0.005 U mL−1, respectively. This method was used in the determination of real serum samples, and the results were consistent with that from ELISA. This assay showed three orders of magnitude of sensitivity higher than that of ELISA, which provided promising simultaneous multianalyte immunoassays (SMIAs) in the early diagnosis of pancreatic cancer. |
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