Chemiluminescence

In electrochemiluminescence, ruthenium chelate attached to a biological substance, when stimulated by applied potential, emits light at a particular wavelength due to a redox reaction to signal the presence of a bioanalyte. This ORIGEN™ technology is a trade mark of IGEN International and has developed a multichannel detection platform. Chemiluminescence in biological systems utilizes the firefly luciferase system. Luciferase catalyzes the reaction of luciferin with ATP, resulting in the emission of photons. The half life of the chemilumines-cence has been progressively increased from a flash (seconds) to a glow (hours) luminescent signal, enabling the development of homogeneous HTS assays.

A. Electrochemiluminescence

Electrochemiluminescence (ECL) assay technology developed by IGEN is marketed as ORIGEN technology. ECL utilizes stable ruthenium chelate (TAG) which in the presence of tripropylamine participates in a luminescent reaction that is triggered by the application of low voltage [56]. ECL is capable of quanti-tating the specific binding of two molecules. In ECL, light is generated when low voltage is applied to an electrode, triggering a cyclical oxidation-reduction reaction of the ruthenium metal ion (Ru2+). The Ru2+ is bound in a chelate of tris-(bipyridine). Tripropylamine (TPA) present in molar excess is the second reaction component that is consumed in the oxidation process, recycling the ruthenium chelate (Fig. 16A). The tracer molecule, Ru-chelate, is conjugated to the antibody. The Ru2+ labeled component is captured on the surface of polystyrene magnetic beads and brought to the surface of an electrode by applying a magnetic field through a movable magnet. TPA is introduced into the flow cell and a voltage is applied that oxidizes both ruthenium and TPA simultaneously (Fig. 16B). TPA by losing a proton becomes a reducing agent and transfers electrons to the ruthenium ions in an excited state and then decays to the ground state, releasing a photon in the process, which is quantitated at 620 nm [56,57]. The reduced Ru2+ is recycled, which along with TPA will intensify and amplify the ECL signal and is read in the ORIGEN® analyzer.

1. Instrumentation

The ORIGEN® 1.5 analyzer by IGEN® International, Inc., for analytical assays and the ORIGEN® ECLM8 electrochemiluminescent reader with 8 ECL modules

Figure 16 Electrochemiluminescence assay technology. (A) Structure of ruthenium (II)tris-(bipyridine)NHS ester. (B) Schematic representation of electrochemiluminescence reaction. (C) Schematic representation of ECL process. (Courtesy of IGEN, Inc.)

Ru(bpy)3

TPA*

TPA*

Ru(bpy)3

TPA*

TPA = Tripropylamine

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