Gold-coated magnetic particles for solid-phase immunoassays: enhancing immobilized antibody binding efficiency and analytical performance.
The preparation and characterization of gold-coated magnetic particles are described for use as more efficient solid-phase materials in immunoassay development. A thin gold coating on commercial tosylated magnetic polystyrene particles (4.5 microm) is achieved via an electroless plating method involving initial reaction of the particles with Sn(II), followed by redox deposition of Ag0, that serves as a catalytic site for the subsequent reduction of Na3Au(SO3)2 in the presence of formaldehyde to yield the adhered gold layer. Scanning electron microscopy, energy-dispersive X-ray analysis, and X-ray photoelectron spectroscopy indicate the presence of the desired Au0 outer layer. To characterize the improved yield of antibody binding sites on such gold-coated phases, the modified particles are reacted with the free thiols of Fab' fragments of an anti-alkaline phosphatase (ALP) antibody to orient all the antigenic binding sites in a favorable direction. After equilibration with ALP, the amount of ALP bound to the surface of such particles is nearly 2.5-fold greater than on non-gold-coated particles possessing the same amount of immobilized anti-ALP Fab', but oriented randomly on the surface. The new gold-coated magnetic particles are further used as a solid phase for developing a sandwich-type enzyme immunoassay to detect C-reactive protein (CRP) using horseradish peroxidase as the enzyme label. The gold-coated magnetic particles with anti-CRP monoclonal Fab' reagents provide assays with enhanced assay slope (1.8-fold), lower nonspecific adsorption, and a detection limit improvement of nearly 10-fold (0.14 vs 1.9 ng/mL) compared to the same Fab' anti-CRP immobilized on the initial tosylated polystyrene magnetic particles. The improved assay performance is attributed to the more favorable binding orientation of the self-assembled monolayer of Fab' fragments on the gold-coated particles compared to the random orientation on the non-gold-coated surfaces.
Zhang
Hairong
H
Department of Chemistry, The University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055, USA.
Meyerhoff
Mark E
ME
D000469:Alkaline Phosphatase / Q000737:chemistry*; D002097:C-Reactive Protein / Q000032:analysis*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D008863:Microspheres
D004800:Enzymes, Immobilized; D007140:Immunoglobulin Fab Fragments; D006046:Gold; D002097:C-Reactive Protein; D000469:Alkaline Phosphatase