Milica Sentic

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.

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·mL–1 and 10 μg·mL–1 and a detection limit of 0.5 ng·mL–1. 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.

Mapping electrogenerated chemiluminescence reactivity in space: mechanistic insight into model systems used in immunoassays

The remarkable characteristics of electrogenerated chemiluminescence (ECL) as a readout method are successfully exploited in numerous microbead-based immunoassays. However there is still a lack of understanding of the extremely high sensitivity of such ECL bioassays. Here the mechanisms of the reaction of the Ru(bpy)32+ luminophore with two efficient co-reactants (TPrA or DBAE) were investigated by mapping the ECL reactivity at the level of single Ru(bpy)32+-functionalized beads. Micrometric non-conductive beads were decorated with the ruthenium label via a sandwich immunoassay or via a peptide bond. Mapping the ECL reactivity on one bead demonstrates the generation of the excited state at a micrometric distance from the electrode by reaction of surface-confined Ru(bpy)32+ with diffusing TPrA radicals. The signature of the TPA˙+ lifetime is obtained from the ECL profile. Unlike the reaction with Ru(bpy)32+ in solution, DBAE generates very low ECL intensity in the bead-based format suggesting more unstable radical intermediates. The 3D imaging approach provides insights into the ECL mechanistic route operating in bioassays and on the optical effects that focus the ECL emission.

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)32+ 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.

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 (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.

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.

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.