Preparation, Characterization And Applications Of SPA-PAMAM-Au Nanocomposites

Immuno-gold staining (IGS) which take advantage of the excellent properties of gold nanoparticles has been widely used in bioanalyses, in which some specific protein is used to warp colloidal gold for detecting the protein's target. The problems and shortages of IGS appear gradually along with enlargement of the marking area and requirement in improvement of detecting precision. First, the preparation of colloidal gold particles requires the equipment be very clean and the control of the size and distribution of the very small size of the gold particles is difficult. Second, the mechanism of protein adsorbing on the surface of colloidal gold is not completely elucidated at present. So it is necessary to study some new system by which the gold nanoparticles synthesized can be meet the requirements cited above. Such a system in this paper was chosen NH₂-terminated polyamidoamine (PAMAM), a water-soluble dendrimer which can adsorb on protein and the adsorbing mechanism is well elucidated to make the size of gold nanoparticles controllable. Staphylococcus aureus protein A (SPA) with excellent immunological properties was used to wrap the PAMAM-Au for preparing SPA-PAMAM-Au nanocomposites.This paper mainly included three parts, i.e., 1) synthesis and purification, and characterization of NH₂-terminated PAMAM; 2) synthesis and characterization of SPA-PAMAM-Au nanocomposites; and 3) staining assay of a typical antigen by SPA -PAMAM-Au nanocomposites. The main results are as following.Firstly, generation 2.0 PAMAM (G2.0 PAMAM) was synthesized in a relatively simple procedure. Structural characterization showed that the G2.0 PAMAM and its precursors self-synthesized have the same basic structural characters to the data reported. Chromatographic analyses showed some unnecessary components existing in the G2.0 PAMAM synthesized, which yet can basically be purified by further column chromatographic separation. Secondly, the SPA-PAMAM-Au nanocomposites were synthesized by simply adding SPA into the PAMAM ( G2.0) aqueous solution where reducing HAuCl₄ into Au⁰ with NaBH₄ at the same time. Characterization of the nanocomposites showed that the nanocomposites have a typical size of about 3nm and very narrow size-distribution, and were water-soluble and able to combine well with some specific antibody. The study on the solution chemistry of the formation process of the nanocomposites showed that pH is an important influencing factor, the least amount of protein to wrap PAMAM-Au is acceptable in terms of both the sensitivity and cost of the nanocomposites system. The study of optical properties of the nanocomposites showed that SPA-PAMAM-Au nanocomposites system emitted obvious bluish luminescence after excited. These specific optical properties make the nanocomposites an prosperous potential in being as an excellent optical marker.Finally, the application of the SPA-PAMAM-Au nanocomposites in Dot Immuno Gold Filtration Assay (DIGFA) to stain Carcinoembryonic antigen showed that the nanocomposites could be detect supersensitively carcinoembryonic antigen. We find this probe is also significant on fluorescence detecting because there is a very apparent change in fluorescence spectra after SPA-PAMAM-Au nanocomposites bind a little of CEA antibody or CEA antibody plus CEA. This suggests that SPA-PAMAM-Au nanocomposites could be used as supersensitive optical probe for bioanalyses and medical diagnoses.