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

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 generation does not suffer from background signals, such as the typical autofluorescence of biological samples. The presented surface-confined ECL microscopy should find promising applications in ultrasensitive single cell imaging assays.

Dual Enzymatic Detection by Bulk Electrogenerated Chemiluminescence.

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 (ß-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.

Wireless Synthesis and Activation of Electrochemiluminescent Thermoresponsive Janus Objects Using Bipolar Electrochemistry.

In this work, bipolar electrochemistry (BPE) is used as a dual wireless tool to generate and to activate a thermoresponsive electrochemiluminescent (ECL) Janus object. For the first time, BPE allows regioselective growth of a poly(N-isopropylacrylamide) (pNIPAM) hydrogel film on one side of a carbon fiber. It is achieved thanks to the local reduction of persulfate ions, which initiate radical polymerization of NIPAM. By controlling the electric field and the time of the bipolar electrochemical reactions, we are able to control the length and the thickness of the deposit. The resulting pNIPAM film is found to be swollen in water at room temperature and collapsed when heated above 32 °C. We further incorporated a covalently attached ruthenium complex luminophore, Ru(bpy)32+, in the hydrogel film. In the second time, BPE is used to activate remotely the electrogenerated chemiluminescence (ECL) of the Ru(bpy)32+ moieties in the film. We take advantage of the film responsiveness to amplify the ECL signal. Upon collapse of the film, the ECL signal, which is sensitive to the distance between adjacent Ru(bpy)32+ centers, is strongly amplified. It is therefore shown that BPE is a versatile tool to generate highly sophisticated materials based on responsive polymers, which could lead to sensitive sensors.

Microscopic imaging and tuning of electrogenerated chemiluminescence with boron-doped diamond nanoelectrode arrays.

Nanoelectrode arrays (NEAs) are increasingly applied for a variety of electroanalytical applications; however, very few studies dealt with the use of NEAs as an electrochemical generator of electrogenerated chemiluminescence (ECL). In the present study, arrays of nanodisc and nanoband electrodes with different dimensions and inter-electrode distances were fabricated by e-beam lithography on a polycarbonate layer deposited on boron-doped diamond (BDD) substrates. In particular, NEAs with 16 different geometries were fabricated on the same BDD sample substrate obtaining a multiple nanoelectrode and ultramicroelectrode array platform (MNEAP). After electrochemical and morphological characterization, the MNEAP was used to capture simultaneously with a single image the characteristic behaviour of ECL emission from all the 16 arrays. Experiments were performed using Ru(bpy)3 (2+) as the ECL luminophore and tri-n-propylamine (TPrA) as the co-reactant. With a relatively limited number of experiments, such an imaging procedure allowed to study the role that geometrical and mechanistic parameters play on ECL generation at NEAs. In particular, at high concentrations of TPrA, well-separated individual ECL spots or bands revealed an ECL signal which forms a pattern matching the nanofabricated structure. The analysis of the imaging data indicated that the thickness of the ECL-emitting zone at each nanoelectrode scales inversely with the co-reactant concentration, while significantly stronger ECL signals were detected for NEAs operating under overlap conditions.

Antagonistic effects leading to turn-on electrochemiluminescence in thermoresponsive hydrogel films.

We report the effects of the swell-to-collapse transition on the electrochemical and electrochemiluminescence (ECL) properties of thermoresponsive pNIPAM films incorporating covalently-attached Ru(bpy)32+ luminophores. Upon the collapse of the film, the number of electrochemically-active Ru(bpy)32+ centers increases, due to the reduced distance between adjacent redox centers. To generate ECL, cationic and anionic coreactants are employed, which are free to diffuse in the medium. In both cases, the ECL intensity exhibits a remarkable amplification of up to 58-fold at the swell-to-collapse transition, whereas the oxidation current undergoes a steep decrease in the case of the cationic coreactant. Indeed, the diffusion of the coreactant is hindered by the collapse of the film. The fact that ECL emission is enhanced whereas less coreactant radicals are generated is an intriguing observation because it is classically believed that the higher the oxidation rate of the coreactant the stronger the ECL emission. Thanks to the study of such films where antagonistic effects occur, we demonstrate that ECL enhancement is neither correlated with the oxidation efficiency of the coreactant nor with the hydrophilic-hydrophobic transition, but solely due to film shrinking. The decrease of the distance between adjacent redox centers, which favors better electron-transfer processes, is found to be the main parameter governing the ECL enhancement. Finally, it is noteworthy that the present turn-on ECL signal with increasing temperature is unique compared to many thermoresponsive luminescent systems.

A Sensitive Electrochemiluminescence Immunosensor for Celiac Disease Diagnosis Based on Nanoelectrode Ensembles.

We report here the design of a novel immunosensor and its application for celiac disease diagnosis, based on an electrogenerated chemiluminescence (ECL) readout, using membrane-templated gold nanoelectrode ensembles (NEEs) as a detection platform. An original sensing strategy is presented by segregating spatially the initial electrochemical reaction and the location of the immobilized biomolecules where ECL is finally emitted. The recognition scaffold is the following: tissue transglutaminase (tTG) is immobilized as a capturing agent on the polycarbonate (PC) surface of the track-etched templating membrane. It captures the target tissue transglutaminase antibody (anti-tTG), and finally allows the immobilization of a streptavidin-modified ruthenium-based ECL label via reaction with a suitable biotinylated secondary antibody. The application of an oxidizing potential in a tri-n-propylamine (TPrA) solution generates an intense and sharp ECL signal, suitable for analytical purposes. Voltammetric and ECL analyses evidenced that the ruthenium complex is not oxidized directly at the surface of the nanoelectrodes; instead ECL is generated following the TPrA oxidation, which produces the TPrA•+ and TPrA• radicals. With NEEs operating under total overlap diffusion conditions, high local fluxes of these reactive radicals are produced by the nanoelectrodes in the immediate vicinity of the ECL labels, so that they efficiently generate the ECL signal. The radicals can diffuse over short distances and react with the Ru(bpy)32+ label. In addition, the ECL emission is obtained by applying a potential of 0.88 V versus Ag/AgCl, which is about 0.3 V lower than when ECL is initiated by the electrochemical oxidation of Ru(bpy)3(2+). The immunosensor provides ECL signals which scale with anti-tTG concentration with a linearity range between 1.5 ng·mL­1 and 10 µg·mL­1 and a detection limit of 0.5 ng·mL­1. The sensor is finally applied to the analysis of anti-tTG in human serum samples, showing to be suitable to discriminate between healthy and celiac patients.

Luminescent Metal-Organic Frameworks for Electrochemiluminescent Detection of Water Pollutants.

The modern lifestyle has increased our utilization of pollutants such as heavy metals, aromatic compounds, and contaminants that are of rising concern, involving pharmaceutical and personal products and other materials that may have an important environmental impact. In particular, the ultimate results of the intense use of highly stable materials, such as heavy metals and chemical restudies, are that they turn into waste materials, which, when discharged, accumulate in environmental water bodies. In this context, the present review presents the application of metal-organic frameworks (MOFs) in electrochemiluminescent (ECL) sensing for water pollutant detection. MOF composites applied as innovative luminophore or luminophore carriers, materials for electrode modification, and the enhancement of co-reaction in ECL sensors have enabled the sensitive monitoring of some of the most common contaminants of emerging concern such as heavy metals, volatile organic compounds, pharmaceuticals, industrial chemicals, and cyanotoxins. Moreover, we provide future trends and prospects associated with ECL MOF composites for environmental sensing.

Light-emitting electrochemical "swimmers".

Swimmer in the dark: propulsion of a conducting object is intrinsically coupled with light emission using bipolar electrochemistry. Asymmetric redox activity on the surface of the swimmer (black bead) causes production of gas bubbles to propel the swimmer in a glass tube with simultaneous electrochemiluminescence (ECL) emission to monitor the progress of the swimmer.

3D electrogenerated chemiluminescence: from surface-confined reactions to bulk emission.

Among luminescence techniques, electrogenerated chemiluminescence (ECL) provides a unique level of manipulation of the luminescent process by controlling the electrochemical trigger. Despite its attractiveness, ECL is by essence a 2D process where light emission is strictly confined to the electrode surface. To overcome this intrinsic limitation, we added a new spatial dimension to the ECL process by generating 3D ECL at the level of millions of micro-emitters dispersed in solution. Each single object is addressed remotely by bipolar electrochemistry and they generate collectively the luminescence in the bulk. Therefore, the entire volume of the solution produces light. To illustrate the generality of this concept, we extended it to a suspension of multi-walled carbon nanotubes where each one acts as an individual ECL nano-emitter. This approach enables a change of paradigm by switching from a surface-limited process to 3D electrogenerated light emission.

Electrochemiluminescent swimmers for dynamic enzymatic sensing.

An electrochemiluminescent (ECL) swimmer driven by bipolar electrochemistry is reported for enzymatic glucose sensing. The chemo-mechanical motion is induced by localized hydrogen bubble generation. The concomitant oxidation of the luminophore and of the enzymatically-produced NADH leads to ECL emission with a direct glucose-dependent light intensity. We demonstrate herein the local sensing and reporting of glucose in a concentration gradient explored by the ECL swimmer. Such a dynamic sensing approach combines in a synergetic way the wireless propulsion with the enzymatic selectivity using ECL as a readout method at the level of moving objects.