Insights into the mechanism of coreactant electrochemiluminescence facilitating enhanced bioanalytical performance

Abstract

Electrochemiluminescence (ECL) is a powerful transduction technique with a leading role in the biosensing field due to its high sensitivity and low background signal. Although the intrinsic analytical strength of ECL depends critically on the overall efficiency of the mechanisms of its generation, studies aimed at enhancing the ECL signal have mostly focused on the investigation of materials, either luminophores or coreactants, while fundamental mechanistic studies are relatively scarce. Here, we discover an unexpected but highly efficient mechanistic path for ECL generation close to the electrode surface (signal enhancement, 128%) using an innovative combination of ECL imaging techniques and electrochemical mapping of radical generation. Our findings, which are also supported by quantum chemical calculations and spin trapping methods, led to the identification of a family of alternative branched amine coreactants, which raises the analytical strength of ECL well beyond that of present state-of-the-art immunoassays, thus creating potential ECL applications in ultrasensitive bioanalysis.

Spatially resolved electrochemiluminescence through a chemical lens

“…It provides high ECL efficiency in heterogeneous as well as homogeneous formats and it is the most widely exploited ECL system to detect various biomolecules (e.g., proteins, peptides, ligands and oligonucleotides) and DNA, for immunological assays and ECL microscopy. [11][12][13][14][15][16][17][18][19][20][21][22] The heterogeneous ECL mechanism of the [Ru(bpy) 3 ] 2+ /TPrA system is an active area of investigation. 14,32,33,43,44 In such a conguration, the ECL luminophore is not free to diffuse and cannot be directly oxidised at the electrode.…”

“…47 Considering the respective micrometric and nanometric dimensions of the bead and of the tunnelling distance, ECL resulting from the direct oxidation of [Ru(bpy) 3 ] 2+ can be neglected. 14,33,45,48 The ECL is induced exclusively by the direct oxidation of the freely diffusing TPrA (eqn (1)) that, upon oxidation, partially undergoes a deprotonation reaction (eqn (2)). Thus, it forms a high energetic radical species 49 able to reduce the ECL luminophore [Ru(bpy) 3 ] 2+ to [Ru(bpy) 3 ] + (eqn (3)).…”

“…46 Many research groups, including ours, mapped the ECL reactivity and the emission spatial distribution in such a context. 14,15,33,43 The maximum of ECL emission occurs in the micrometric region where concentrations of TPrAc and TPrAc + radicals are locally the highest. 43,44 We also demonstrated, by analysing the side-view ECL image of the beads that only luminophores located within the 3 mm region close to the electrode contribute to the signal.…”

“…Noteworthily, from the general equation scheme (eqn (1)-(5)), we recognize the essential role of the proton-accepting base in dening the concentration gradient ratio of [TPrAc]/[TPrAc + ], as described by the pseudo rst order deprotonation reaction (eqn (2)). 14,32,44,50 Therefore, we analysed the ECL emission from Ru@bead with a top-view conguration (Scheme 1) with different phosphate buffer (PB) concentrations ( Fig. 1 and S1 †).…”

“…[6][7][8][9][10][11] Besides the regular analytical applications, the combination of ECL with microscopy (ECLM) is an emerging technique that provides high lateral resolution, typical of microscopy, with surface-conned processes since the emission is restricted within the ECL reaction layer (i.e., mm range near the electrode surface). [12][13][14] Recently, we reported the rst example of ECLM for the visualization of single cells and detection of cancer biomarkers on the cellular membrane. [15][16][17] Different approaches have also been proposed to image cells by ECLM.…”