Rather than [Ru(bpy)3]2+, the light-emitting molecule used in Enhanced Electrochemiluminescence (EECL) is an electronically Neutral Ruthenium Complex (NRC). The differences between [Ru(bpy)3]2+ and NRC lie in electronic neutrality and ligand heterogenity. However, the neutrality and heterogenity do not guarantee brighter ECL, nor do other molecular structural features. This is one reason why the first-in-class [Ru(bpy)3]2+ has remained best-in-class for more than twenty-five years until we made a change.
Under the same conditions, the ECL from NRC can be more than twice as strong as that of [Ru(bpy)3]2+, and the antibodies labeled with NRC also have weaker non-specific adsorption. An immediately obvious way of using NRC would be to raise the signal level and lower the limit of detection. However, the way we take advantage of NRC goes beyond the generally held perception.
Because of the stronger emission from NRC, the incubation time required to reach a signal level equivalent to that of [Ru(bpy)3]2+ can be reduced. Based on the precise control of the incubation and our study on the kinetics of immunocomplex formation, we are able to carry out the incubation over a 5-minute period, which is much shorter than other chemiluminescence assays, without sacrificing measurement precision.
The ECL of both NRC and [Ru(bpy)3]2+ are sensitive to the applied potential, and the background noise is also a function of the potential. However, their sensitivities are different. With the strongest emission and the lowest sensitivity to the potential, NRC enables the ECL to be generated at lower potentials with higher signal-to-noise ratios, while maintaining a signal level same as or similar to that of [Ru(bpy)3]2+. The excellent controllability of the ECL plays an important role in striking the balance between the signal level and the signal-to-noise ratio (or response-dose relation) in our EECL system.