A real-time immuno-PCR assay for the flame retardant tris(2,3-dibromopropyl) isocyanurate using a probe DNA conjugated to gold nanoparticles

We report on an improved real-time immuno-PCR assay for the flame retardand tris(2,3-dibromopropyl) isocyanurate (TBC). For establishing this immunoassay, two kinds of TBC antigen (TBC-OVA and TBC-BSA) were prepared by coupling TBC hapten to ovalbumin and to bovine serum, respectively. The anti-TBC polyclonal antibody was obtained via immunization of rabbits. Subsequently, a bio-functionalized probe was synthesized by conjugating thiol-capped DNA and goat anti-rabbit IgG to gold nanoparticles. This immunoassay involves the follow steps: (a) fixing the coating antigen (TBC-OVA) onto the surface of PCR tubes, and blocking the unbound sites; (b) adding anti-TBC antibody and sample solutions, upon which the TBC analyte in sample and TBC-OVA fixed on the tube well competitively combine with anti-TBC antibody; (c) adding bio-functionalized probe to specifically recognize anti-TBC antibody and release template DNA during PCR amplification. The fluorescence signal was detected via SYBR Green at excitation/emission wavelengths of 497/520 nm. Under optimized conditions, TBC could be detected in the 0.1 pga (TM) L-1 to 0.1 nga (TM) L-1 concentration range, with a detection limit as low as 0.97 pga (TM) L-1. To our knowledge, this immunoassay is the most sensitive one at present and - in our perception - has a large potential for trace detection of TBC in environmental samples.