Nanogold-polyaniline-nanogold microspheres-functionalized molecular tags for sensitive electrochemical immunoassay of thyroid-stimulating hormone

Methods based on nanomaterial labels have been developed for electrochemical immunosensors and immunoassays, but most involved low sensitivity. Herein a novel class of molecular tags, nanogold-polyaniline-nanogold microspheres (GPGs), was first synthesized and functionalized with horseradish peroxidase-conjugated thyroid-stimulating hormone antibody (HRP-Ab(2)) for sensitive electrochemical immunoassay of thyroid-stimulating hormone (TSH). X-ray diffraction, confocal Raman spectroscopy, scanning electron microscope and transmission electron microscope were employed to characterize the prepared GPGs. Based on a sandwich-type immunoassay format, the assay was performed in pH 5.0 acetate buffer containing 6.0 mmol L-1 H2O2 by using GPG-labeled HRP-Ab(2) as molecular tags. Compared with pure polyaniline nanospheres and gold nanoparticles alone, the GPG hybrid nanostructures increased the surface area of the nanomaterials, and enhanced the immobilized amount of HRP-Ab(2). Several labeling protocols comprising HRP-Ab(2), nanogold particle-labeled HRP-Ab(2), and polyaniline nanospheres-labeled HRP-Ab(2), were also investigated for determination of TSH and improved analytical features were obtained by using the GPG-labeled HRP-Ab(2). With the GPG labeling method, the effects of incubation time and pH of acetate buffer on the current responses of the immunosensors were also studied. The strong attachment of HRP-Ab(2) to the GPGs resulted in a good repeatability and intermediate precision down to 7%. The dynamic concentration range spanned from 0.01 to 20 mu IU mL(-1) with a detection limit (LOD) of 0.005 mu IU mL(-1) TSH at the 3s(B) criterion. Significantly, no significant differences at the 0.05 significance level were encountered in the analysis of 15 spiking serum samples between the developed electrochemical immunoassay and the commercially available enzyme-linked immunosorbent assay (ELISA) method for determination of TSH. (C) 2012 Elsevier B.V. All rights reserved.