The combination of enzymes, as recognition elements for specific analytes, and of electrogenerated chemiluminescence (ECL) as a readout method has proven to be a valuable strategy for sensitive and specific analytical detection. However, ECL is intrinsically a 2D process which could potentially limit the analysis of inhomogeneous samples. Here, we show how a bulk ECL signal, generated by thousands of carbon microbeads remotely addressed via bipolar electrochemistry, are implemented as a powerful tool for the concomitant ECL sensing and imaging of two enzymatic substrates. We selected two enzymes (glucose dehydrogenase and choline oxidase) that react with their respective model substrates and produce in situ chemical species (β-nicotinamide adenine dinucleotide (NADH) and H2O2) acting as coreactants for the ECL emission of different luminophores ([Ru(bpy)3](2+) at λ = 620 nm and luminol at λ = 425 nm, respectively). Both enzymes are spatially separated in the same capillary. We demonstrate thus the simultaneous quantitative determination of both glucose and choline over a wide concentration range. The originality of this remote approach is to provide a global chemical view through one single ECL image of inhomogeneous samples such as a biochemical concentration gradient in a capillary configuration. Finally, we report the first proof-of-concept of dual biosensing based on this bulk ECL method for the simultaneous imaging of both enzymatic analytes at distinct wavelengths.
Bipolar electrochemistry (BPE) is exploited here to address simultaneously thousands of carbon microbeads dispersed in solution. The suspension is placed in a capillary between two feeder electrodes generating an electric field in situ. The simultaneous occurrence of electrogenerated chemiluminescence (ECL) at each bead leads to a global 3D luminescent phenomenon, remotely driven by BPE. This concept, demonstrated previously as a proof‐of‐principle, is extended here to luminol in order to tune the emission wavelength. Determinant analytical parameters such as the applied voltage and the number of emitters were studied for the optimization of the system. The linearity of the ECL intensity in the bulk as a function of the luminophore concentration is successfully demonstrated for both ECL systems [luminol and Ru(bpy)32+]. Finally, we show that both luminophores can be combined in the same capillary and addressed by BPE, thus leading to simultaneous ECL emissions at distinct wavelengths.
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