Ternary covalent conjugate (antibody–gold nanoparticle–peroxidase) for signal enhancement in enzyme immunoassay†
A synthesis of a new horseradish peroxidase (HRP) conjugate and antibody covalently bound with gold nanoparticles (GNPs) with a high enzyme content was first developed. The synthesis conditions were optimized by varying the concentrations of the reacting compounds. In the conjugate, which was synthesized under the optimized conditions, one gold nanoparticle carrier (Ø 30 nm) contained 13 active HRP and 1 anti-troponin I antibody molecule at its surface. The applicability of the proposed ternary conjugate was successfully proved in chemiluminescent ELISA by determining human troponin I. The sensitivity of the assay using the ternary conjugate was higher than that of the assay with the streptavidin–biotin system (biotinylated antibody and streptavidin–HRP conjugate). High sensitivity was achieved due to the dual signal amplification from use of a potent enhancer of HRP-catalyzed chemiluminescence (3-(10′-phenothiazinyl)propane-1-sulfonate) and the high HRP content in the conjugate. A replacement of two-step streptavidin–biotin amplification system with the ternary conjugate decreased the assay time. The use of the proposed conjugate significantly reduced antibody and HRP consumption per assay.