Electrogenerated chemiluminescence from luminol-labelled microbeads triggered by in situ generation of hydrogen peroxide.

We developed a sensing strategy that mimics the bead-based electrogenerated chemiluminescence immunoassay. However, instead of the most common metal complexes, such as Ru or Ir, the luminophore is luminol. The electrogenerated chemiluminescence of luminol was promoted by in situ electrochemical generation of hydrogen peroxide at a boron-doped diamond electrode. The electrochemical production of hydrogen peroxide was achieved in a carbonate solution by an oxidation reaction, while at the same time, microbeads labelled with luminol were deposited on the electrode surface. For the first time, we proved that was possible to obtain light emission from luminol without its direct oxidation at the electrode. This new emission mechanism is obtained at higher potentials than the usual luminol electrogenerated chemiluminescence at 0.3-0.5 V, in conjunction with hydrogen peroxide production on boron-doped diamond at around 2-2.5 V (vs Ag/AgCl).

Redox-mediated electrochemiluminescence enhancement for bead-based immunoassay.

Electrochemiluminescence (ECL) is a highly sensitive mode of detection utilised in commercialised bead-based immunoassays. Recently, the introduction of a freely diffusing water-soluble Ir(iii) complex was demonstrated to enhance the ECL emission of [Ru(bpy)3]2+ labels anchored to microbeads, but a comprehensive investigation of the proposed 'redox-mediated' mechanism was not carried out. In this work, we select three different water-soluble Ir(iii) complexes by virtue of their photophysical and electrochemical properties in comparison with those of the [Ru(bpy)3]2+ luminophore and the TPrA co-reactant. A systematic investigation of the influence of each Ir(iii) complex on the emission of the Ru(ii) labels on single beads by ECL microscopy revealed that the heterogeneous ECL can be finely tuned and either enhanced up to 107% or lowered by 75%. The variation of the [Ru(bpy)3]2+ ECL emission was correlated to the properties of each Ir(iii)-based mediator, which enabled us to decipher the mechanism of interaction and define guidelines for the future design of novel Ir(iii) complexes to further enhance the ECL emission of bead-based immunoassays. Ultimately, we showcase the potential of this technology for practical sample analysis in commercial instruments by assessing the enhancement of the collective ECL intensity from a bead-based system.

Boron-Doped Diamond Electrode Outperforms the State-of-the-Art Electrochemiluminescence from Microbeads Immunoassay.

Electrochemiluminescence (ECL) is a powerful transduction technique where light emission from a molecular species is triggered by an electrochemical reaction. Application to biosensors has led to a wide range of electroanalytical methods with particular impact on clinical analysis for diagnostic and therapeutic monitoring. Therefore, the quest for increasing the sensitivity while maintaining reproducible and easy procedures has brought investigations and innovations in (i) electrode materials, (ii) luminophores, and (iii) reagents. Particularly, the ECL signal is strongly affected by the electrode material and its surface modification during the ECL experiments. Here, we exploit boron-doped diamond (BDD) as an electrode material in microbead-based ECL immunoassay to be compared with the approach used in commercial instrumentation. We conducted a careful characterization of ECL signals from a tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)32+)/tri-n-propylamine (TPrA) system, both homogeneous (i.e., free diffusing Ru(bpy)32+) and heterogeneous (i.e., Ru(bpy)32+ bound on microbeads). We investigated the methods to promote TPrA oxidation, which led to the enhancement of ECL intensity, and the results revealed that the BDD surface properties greatly affect the ECL emission, so it does the addition of neutral, cationic, or anionic surfactants. Our results from homogeneous and heterogeneous microbead-based ECL show opposite outcomes, which have practical consequences in ECL optimization. In conclusion, by using Ru(bpy)32+-labeled immunoglobulins bound on microbeads, the ECL resulted in an increase of 70% and a double signal-to-noise ratio compared to platinum electrodes, which are actually used in commercial instrumentation for clinical analysis. This research infers that microbead-based ECL immunoassays with a higher sensitivity can be realized by BDD.