Zhang Hairong, Meyerhoff Mark E
Department of Chemistry, The University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055, USA.
Anal Chem. 2006 Jan 15;78(2):609-16. doi: 10.1021/ac051720x.
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.
本文描述了用于免疫分析开发中更高效固相材料的金包覆磁性颗粒的制备与表征。通过化学镀法在商用甲苯磺酰化磁性聚苯乙烯颗粒（4.5微米）上形成薄金涂层，该方法包括颗粒先与Sn(II)反应，随后进行Ag0的氧化还原沉积，Ag0作为催化位点，在甲醛存在下将Na3Au(SO3)2还原，从而形成附着的金层。扫描电子显微镜、能量色散X射线分析和X射线光电子能谱表明存在所需的Au0外层。为了表征此类金包覆相上抗体结合位点的产率提高情况，将改性颗粒与抗碱性磷酸酶（ALP）抗体的Fab'片段的游离巯基反应，以使所有抗原结合位点沿有利方向排列。与ALP平衡后，结合到此类颗粒表面的ALP量比具有相同固定化抗ALP Fab'但随机排列在表面的非金包覆颗粒上的量高出近2.5倍。新型金包覆磁性颗粒还用作固相，开发一种夹心型酶免疫分析，以辣根过氧化物酶作为酶标记物检测C反应蛋白（CRP）。与固定在初始甲苯磺酰化聚苯乙烯磁性颗粒上的相同Fab'抗CRP相比，带有抗CRP单克隆Fab'试剂的金包覆磁性颗粒提供的分析具有更高的分析斜率（1.8倍）、更低的非特异性吸附以及检测限提高近10倍（0.14对1.9纳克/毫升）。分析性能的提高归因于与非金包覆表面上的随机排列相比，Fab'片段自组装单层在金包覆颗粒上具有更有利的结合取向。
