Electrogenerated Chemiluminescence. 77. DNA Hybridization Detection at High Amplification with [Ru(bpy)₃]²⁺-Containing Microspheres

An ultrasensitive DNA hybridization detection method based on electrogenerated chemiluminescence (ECL) using polystyrene microspheres/beads (PSB) as the carrier of the ECL labels, namely, tris(2,2‘-bipyridyl)ruthenium(II) tetrakis(pentafluorophenyl)borate (Ru(bpy)₃[B(C₆F₅)₄]₂), is reported. Probe single-stranded DNA (p-ssDNA) was attached to the surface of magnetic beads (MB) and hybridized with target-ssDNA (t-ssDNA) with immobilized PSB containing a large number of water insoluble Ru(bpy)₃[B(C₆F₅)₄]₂ species (∼7.5 × 10⁹ molecules/bead). With this approach a large amplification factor of Ru(bpy)₃[B(C₆F₅)₄]₂ molecules for each t-ssDNA can be achieved, when each PSB is attached to a limited number of t-ssDNA. The p-ssDNA−MB ↔ t-ssDNA−PSB/Ru(bpy)₃²⁺ conjugates formed were magnetically separated from the reaction media and dissolved in MeCN containing tri-n-propylamine (TPrA) as an ECL coreactant. ECL was produced with a potential scan from 0 to 3.0 V versus Ag/Ag⁺, and the integrated ECL intensity was found to be linearly proportional to the t-ssDNA concentration in a range of 1.0 fM to 10 nM under optimized conditions. ECL signals associated with two base pair mismatched ssDNA and noncomplementary ssDNA can be distinguished well from the ECL signal related to the complementary DNA hybridization. A Poisson distribution is followed when a large number of MB reacts with PSB, and the minimum number of 1.0- and 2.8-μm diameter MB required to bind and magnetically separate a single 10-μm diameter PSB from the reaction solution was estimated to be three and one, respectively. The principle described in this paper could be also applied to many other ECL analyses, such as immunoassays.