Electrogenerated Chemiluminescence. 80. C-Reactive Protein Determination at High Amplification with [Ru(bpy)3]2+-Containing Microspheres

Abstract

Biotinylated anti-C-reactive protein (CRP) species were attached to the surface of streptavidin-coated magnetic beads (MB) and avidin-coated polystyrene microspheres/beads (PSB) entrapping a large number of electrogenerated chemiluminescence (ECL) labels (∼10⁹ Ru(bpy)₃[B(C₆F₅)₄]₂/bead) to form anti-CRP↔MB and Ru(II)⊂PSB/avidin↔anti-CRP conjugates, respectively. Sandwich-type Ru(II)⊂PSB/avidin↔anti-CRP 〈CRP〉 anti-CRP↔MB aggregates were formed when Ru(II)⊂PSB/avidin↔anti-CRP was mixed with anti-CRP↔MB conjugates in the presence of analyte CRP. The newly formed aggregates were magnetically separated from the reaction media and dissolved in MeCN containing tri-n-propylamine as an ECL coreactant. ECL was carried out with a potential scan from 0 to 2.8 V vs Ag/Ag⁺, and the ECL intensity was found to be proportional to the analyte CRP concentration over the range of 0.010−10 μg/mL. The CRP concentration of an unknown human plasma specimen was measured by the standard addition method based on this technique. Elimination of the nonspecific adsorption of the CRP system with several different blocking agents was also studied, and 2.0% bovine serum albumin was found to be best.