Gold/conducting polymer coatings for solid phase immunoassays

Gold coated magnetic particles (MPs) and microtiter plates were developed as novel solid phases for immobilizing antibodies and their fragments (Fab') with improved orientation control and immobilization efficiency for use in immunoassays. An electroless gold plating procedure was adapted to coat polymeric MPs and microtiter plate wells with a thin layer (∼7 nm) of gold. Whole immunoglobulin G antibodies were digested and the resulting Fab' fragments were separated and characterized. The prepared Fab' fragments were immobilized onto the gold coated solid phases through gold-thiolate bonding and their orientation influence on the fragments was evaluated through comparative tests with uncoated solid phases possessing the same amount of immobilized antibody fragments, but oriented randomly on the surface. This resulted in a greater than 2-fold increase in model antigen binding capacity on gold coated solid phases than on uncoated solid phases. A sandwich immunoassay for C-reactive protein (CRP) based on gold coated MPs exhibited an enhanced assay slope (1.8 fold), lower non-specific adsorption and a detection limit improvement of nearly 10 fold (0.14 vs. 1.9 ng/ml) compared to assays carried out with non-gold coated MPs; while the sandwich assay based on gold coated microtiter plates showed a marginal increase in assay slope (13%), due to the limited accessibility of antibodies to surface sites on the gold coated wells. Sandwich assays of CRP and B. Globigii spores based on whole antibodies immobilized on gold coated microtiter plate wells demonstrated increased antibody immobilization efficiency up to 100% due to the increased surface area of gold coating.; A new conductimetric immunosensing system based on conducting polymer coated MPs was also investigated. The sensing system was designed using a microsample chip with interdigitated electrodes and electromagnetic sample collection system. Polypyrrole and poly(3,4-ethylenedioxythiophene) (PEDOT) coated MPs were developed and characterized. Preliminary studies based on PPy coated MPs indicated a dose-response of conductivity in analyzing CRP with the proposed conductimetric immunosensing system. The problems associated with conductivity stability, reproducibility and signal dynamic range were examined. PEDOT coated MPs, which exhibited stable and high conductivity, are proposed to be a more promising candidate for a "proof-of-concept" conductimetric immunosensing system.