Electrochemiluminescence (ECL) is a highly successful technique used in commercial immunoassays for clinical diagnosis. Developing an ECL-based multiplex immunoassay, with the potential to enable high-throughput detection of multiple biomarkers simultaneously, remains a current research interest yet is limited by a narrow choice of ECL luminophores. Herein we report the synthesis, photophysics, electrochemistry, and ECL of several new ruthenium(II) and iridium(III) complexes, three of which are eventually used as signal reporters for multiplex immunoassay. The ECL behaviors of individual luminophores and their mixtures were investigated in multiple modes, including light intensity, spectrum, and image measurements. The spectral peak separation between Ru(bpy)2(dvbpy)2+ (bpy = 2,2'-bipyridine, dvbpy = 4,4'-bis(4-vinylphenyl)-2,2'-bipyridine), and Ir(dFCF3ppy)2(dtbbpy)+ (dFCF3ppy = 3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridinyl]phenyl, dtbbpy = 4,4'-bis( tert-butyl)-2,2'-bipyridine) was up to 145 nm, thus providing the spectrum-resolved possibility of identifying light signals. The potential-resolved ECL signals were achieved for the mixtures of Ir(ppy)3 (ppy = 2-phenylpyridine) with either Ru(bpy)2(dvbpy)2+ or Ir(dFCF3ppy)2(dtbbpy)+, due to the self-annihilation ECL of Ir(ppy)3 at higher potentials, as confirmed by electrochemistry-coupled mass spectrometry. A multiplex immunoassay free of spatial spotting antibodies on plates or substrates was ultimately devised by combining luminophore-loaded polymer beads with the homogeneous sandwich immunoreaction. Using potential and spectrum dual-resolved ECL as the readout signal, simultaneous recognition of three antigens, namely, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and beta-human chorionic gonadotropin (β-HCG), was demonstrated in a single run for a sample volume of 300 μL. These results contribute to the understanding of ECL generation by multiple luminophores and devising spot-free multiplex immunoassays with less sample consumption.

Potential-Resolved Multicolor Electrochemiluminescence for Multiplex Immunoassay in a Single Sample.

We report here the development of coreactant-based electrogenerated chemiluminescence (ECL) as a surface-confined microscopy to image single cells and their membrane proteins. Labeling the entire cell membrane allows one to demonstrate that, by contrast with fluorescence, ECL emission is only detected from fluorophores located in the immediate vicinity of the electrode surface (i.e., 1–2 μm). Then, to present the potential diagnostic applications of our approach, we selected carbon nanotubes (CNT)-based inkjet-printed disposable electrodes for the direct ECL imaging of a labeled plasma receptor overexpressed on tumor cells. The ECL fluorophore was linked to an antibody and enabled to localize the ECL generation on the cancer cell membrane in close proximity to the electrode surface. Such a result is intrinsically associated with the unique ECL mechanism and is rationalized by considering the limited lifetimes of the electrogenerated coreactant radicals. The electrochemical stimulus used for luminescence g...

Single Cell Electrochemiluminescence Imaging: From the Proof-of-Concept to Disposable Device-Based Analysis

https://pubs.rsc.org/en/Content/ArticlePDF/2016/GC/C6GC01101B

Metal-free, visible-light-mediated, decarboxylative alkylation of biomass-derived compounds

Cell-matrix adhesions play essential roles in a variety of biological processes. Herein, we report a label-free method to map cell-matrix adhesions of single living cells on an electrode surface by electrochemiluminescence (ECL). An indium tin oxide electrode modified with a silica nanochannel membrane was used as the substrate electrode, at which the ECL generation from freely diffusing luminophores provided a distinct visual contrast between adhesion sites and noncontacted domains, thus selectively revealing the former in a label-free manner. With this methodology, we studied the spatial distribution, as well as dynamic variation, of cell-matrix adhesions and the adhesion strength at the subcellular level. Cell-matrix adhesions of an advancing cell sheet were finally imaged to study the movement of cells in collective migration. A statistical analysis suggests that cells on the far side of leading edge also have the propensity to migrate and do not act as just passive followers.

Imaging Cell‐Matrix Adhesions and Collective Migration of Living Cells by Electrochemiluminescence Microscopy

The phenomenon of electrochemiluminescence (ECL) is luminescence triggered by electrochemical reactions at electrodes. Heterogeneous electron transfers are deeply dependent on electrode materials, therefore, besides the usual parameters, the right choice of the electrode is a crucial point to address properly in order to maximize the emission efficiency. Many different electrodes have been studied, from metallic -platinum or gold- through transparent indium-doped tin oxide to carbon based electrodes, and others, such as paper based and boron doped diamond. This review summarizes results of ECL at different electrode surfaces, disclosing the relative advantages and disadvantages, with a particular attention to the reference Ru(bpy)32+/TPrA coreactant system. In other words, we offer an insight to recognize and select the optimal electrode material for the ECL systems of interest with particular emphasis on the biosensing applications.

Essential Role of Electrode Materials in Electrochemiluminescence Applications

We demonstrate a new approach to manipulate the selective emission in mixed electrogenerated chemiluminescence (ECL) systems, where subtle changes in co-reactant properties are exploited to control the relative electron-transfer processes of excitation and quenching. Two closely related tertiary-amine co-reactants, tri-n-propylamine and N,N-diisopropylethylamine, generate remarkably different emission profiles: one provides distinct green and red ECL from [Ir(ppy)3] (ppy=2-phenylpyridinato-C2,N) and a [Ru(bpy)3]2+ (bpy=2,2′-bipyridine) derivative at different applied potentials, whereas the other generates both emissions simultaneously across a wide potential range. These phenomena can be rationalized through the relative exergonicities of electron-transfer quenching of the excited states, in conjunction with the change in concentration of the quenchers over the applied potential range.

Control of Excitation and Quenching in Multi‐colour Electrogenerated Chemiluminescence Systems through Choice of Co‐reactant

A series of aliphatic tertiary amines (HEPES, POPSO, EPPS and BIS-TRIS) commonly used to buffer the pH in biological experiments, were examined as alternative, non-toxic co-reactants for the electrogenerated chemiluminescence (ECL) of tris(2,2'-bipyridine)ruthenium(ii) ([Ru(bpy)3](2+)). These were found to be very attractive as "multi-tasking" reagents, serving not only as co-reactants, but also fulfiling the roles of pH buffer and supporting electrolyte within an aqueous environment; thus significantly simplifying the overall ECL analysis. Sub-nanomolar detection limits were obtained for [Ru(bpy)3](2+) in the presence of BIS-TRIS, making this species an valuable option for co-reactant ECL-based bioanalytical applications.

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Electrogenerated chemiluminescence of tris(2,2′ bipyridine)ruthenium(II) using common biological buffers as co-reactant, pH buffer and supporting electrolyte

Noah Kebede

Papers

Control of Excitation and Quenching in Multi‐colour Electrogenerated Chemiluminescence Systems through Choice of Co‐reactant

Electrogenerated chemiluminescence of tris(2,2′ bipyridine)ruthenium(II) using common biological buffers as co-reactant, pH buffer and supporting electrolyte