RESUMO
Visual electrochemiluminescence (ECL) emission from L012 and hydrogen peroxide is generated from an all-solid-state electrochemical cell with a polyacrylamide hydrogel as the solid electrolyte. The emission is strong enough to be visualized with the naked eye, which offers a new idea for the design of an all-solid-state ECL based sensor in air.
RESUMO
The electrochemical visualization of proteins in the plasma membrane of single fixed cells was achieved with a spatial resolution of 160 nm using scanning electrochemical cell microscopy. The model protein, the carcinoembryonic antigen (CEA), is linked with a ruthenium complex (Ru(bpy)32+)-tagged antibody, which exhibits redox peaks in its cyclic voltammetry curves after a nanopipette tip contacts the cellular membrane. Based on the potential-resolved oxidation or reduction currents, an uneven distribution of membrane CEAs on the cells is electrochemically visualized, which could only be achieved previously using super-resolution optical microscopy. Compared with current electrochemical microscopy, the single-cell scanning electrochemical cell microscopy (SECCM) strategy not only improves the spatial resolution but also utilizes the potential-resolved current from the antibody-antigen complex to increase electrochemical imaging accuracy. Eventually, the electrochemical visualization of cellular proteins at the nanoscale enables the super-resolution study of cells to provide more biological information.
Assuntos
Proteínas de Membrana , Microscopia , Microscopia/métodos , Oxirredução , Membrana Celular , Microscopia Eletroquímica de VarreduraRESUMO
As an electrochemical technique offering an optical readout, electrochemiluminescence (ECL) evolved recently into a powerful microscopy technique with the visualization of a wide range of microscopic entities. However, the dynamic imaging of transient ECL events did not receive intensive attention due to the limited number of electrogenerated photons. Here, the reaction kinetics of the model ECL bioassay system was revealed by dynamic imaging of single [Ru(bpy)3]2+-functionalized beads in the presence of the efficient tripropylamine coreactant. The time profile behavior of ECL emission, the variations of the ECL layer thickness, and the position of maximum ECL intensity over time were investigated, which were not achieved by static imaging in previous studies. Moreover, the dynamics of the ECL emission were confronted with the simulation. The reported dynamic ECL imaging allows the investigation of the ECL kinetics and mechanisms operating in bioassays and cell microscopy.
Assuntos
Luminescência , Fotometria , Medições Luminescentes , Microscopia , Técnicas Eletroquímicas/métodosRESUMO
Electrochemiluminescence (ECL) microscopy is successfully applied to image cells, micro-/nano-objects, and electrochemical processes at electrode surfaces. The classic ECL tandem system is composed of the [Ru(bpy)3]2+ luminophore with the very efficient tripropylamine (TPA) coreactant. The dramatic decrease of the ECL signal observed when recording successive ECL images constitutes a key limitation for the development of ECL microscopy. Herein, we investigated the progressive decrease of the ECL signal of Chinese hamster ovary (CHO) cells. The plasma membranes of CHO cells were labeled with a [Ru(bpy)3]2+ derivative, and the ECL images were recorded using the TPA coreactant. We demonstrate that the loss of the ECL signal is related to the electrochemical step because of a progressive lower TPA oxidation current. We tested a cathodic regenerative treatment of the electrode surface, which allowed us to restore the initial TPA oxidation intensity and thus to record a sequence of ECL images without any vanishing of the light signal. The electrochemical approach presented here is an essential step for the development of ECL microscopy of cells.
Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Medições Luminescentes , Animais , Células CHO , Células Cultivadas , Cricetulus , Propriedades de SuperfícieRESUMO
The effects of photobleaching on electrochemiluminescence (ECL) was investigated for the first time. The plasma membrane of Chinese Hamster Ovary (CHO) cells was labeled with a [Ru(bpy)3 ]2+ derivative. Selected regions of the fixed cells were photobleached using the confocal mode with sequential stepwise illumination or cumulatively and they were imaged by both ECL and photoluminescence (PL). ECL was generated with a model sacrificial coreactant, tri-n-propylamine. ECL microscopy of the photobleached regions shows lower ECL emission. We demonstrate a linear correlation between the ECL decrease and the PL loss due to the photobleaching of the labels immobilized on the CHO membranes. The presented strategy provides valuable information on the fundamentals of the ECL excited state and opens new opportunities for exploring cellular membranes by combining ECL microscopy with photobleaching techniques such as fluorescence recovery after photobleaching (FRAP) or fluorescence loss in photobleaching (FLIP) methods.
Assuntos
Membrana Celular/química , Corantes Fluorescentes/química , Compostos Organometálicos/química , Animais , Técnicas Biossensoriais , Células CHO , Membrana Celular/ultraestrutura , Cricetulus , Técnicas Eletroquímicas , Medições Luminescentes , Microscopia Confocal , Fotodegradação , Propilaminas/químicaRESUMO
Here, a microelectrode approach is established to measure the flip-flop rate of cholesterol in plasma membranes at single living cells. The initial validation is performed in a modeled phospholipid bilayer positioned at an interconnecting hole between two compartments, in which cholesterol in one compartment diffuses into the other one through a flip-flop movement in the bilayer and is then detected by a cholesterol oxidase-modified microelectrode. As compared with the time (140 ± 28 s) for free cholesterol transport in absence of the bilayer, a prolonged time (702 ± 42 s) is needed to observe the current increase in the presence of the bilayer. The difference in the time (562 s) gives the estimated flip-flop time of cholesterol in the bilayer. The position of the microelectrode in contact with a living cell and the injection of cholesterol inside the cell are further applied to measure the cholesterol flip-flop in the plasma membrane. The average time (1183 ± 146 s) is obtained to observe an additional current increase at the microelectrode, which reflects the cholesterol flip-flop rate in plasma membranes in single living cells. All these results support the establishment of this microelectrode approach for the study of the cholesterol flip-flop process in lipid membranes.
Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Técnicas Eletroquímicas/métodos , Animais , Colesterol Oxidase/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Peixes , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Oócitos/metabolismo , Fosfatidilcolinas/química , Análise de Célula Única/instrumentação , Análise de Célula Única/métodosRESUMO
Quantification of multiple lipids with different contents in plasma membrane in single cells is significant, but challenging, for investigating lipid interactions and the role of dominant protein transporters. In this paper, comonitoring the alteration of low-content sphingomyelin (SM) and high-content cholesterol in plasma membrane of one living cell is realized by use of luminol electrochemiluminescence (ECL) for the first time. Concentrations of SM as low as 0.5 µM are detected, which permits the measurement of low-content membrane SM in single cells. More membrane cholesterol is observed in individual cells after depletion of membrane SM, providing direct evidence about SM-depletion-induced cholesterol efflux. The upregulation of ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1) in SM-depleted cells induces a further increase in membrane cholesterol. These results imply that higher expression of ABCA1/G1 promotes cholesterol trafficking, which offers additional information to solve the debate about ABC transporters in cholesterol efflux. Moreover, the established approach offers a special strategy to investigate the correlation of membrane lipids and the role of transporters in cholesterol trafficking.
Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Esfingomielinas/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Camundongos , Células RAW 264.7 , Análise de Célula Única , Fatores de TempoRESUMO
In this paper, a titration-type assay is described that determines the minimum concentration of cholesterol in solution that is required to drive net influx of cholesterol to the plasma membrane and thus increase the cholesterol concentration. The increase in cholesterol in the plasma membrane is detected by cholesterol diffusion at the site of contact by a cholesterol oxidase-modified microelectrode. In the presented thermodynamic model, the minimum solution phase cholesterol concentration that drives influx to the plasma membrane is a close approximation of the true solution-phase equilibrium concentration of cholesterol produced from cellular cholesterol efflux and as such it is a quantitative measure of the chemical potential of cholesterol in the cellular plasma membrane. This assay provides a measure of cholesterol chemical potential in the living cellular plasma membrane through reference to a solution concentration which avoids invoking classic kinetic theory to relate a rate to a specific thermodynamic activity and which avoids uncertainty associated with mass transfer phenomena.
Assuntos
Membrana Celular/química , Colesterol/análise , Neurônios/citologia , Membrana Celular/metabolismo , Colesterol/metabolismo , Eletrodos , Humanos , Análise de Célula Única , Termodinâmica , Água/químicaRESUMO
The photoluminescence (PL) of nonthiolate ligand capped Au nanoclusters (NCs) is usually quenched by thiols due to the tight adsorption of thiols to the Au surface and formation of larger non-PL species. However, we here report an unexpected PL enhancement of cytidine stabilized Au (AuCyt) NCs triggered by thiols, such as reduced glutathione (GSH) at sub-µM level, while such phenomena have not been observed for Au NCs capped with similar adenosine/cytidine nucleotides. The mass spectroscopic results indicate that this enhancement may be caused by the formation of smaller, but highly fluorescent, Au species etched by thiols. This enables the sensitive detection of GSH from 20 nM to 3 µM, with an ultralow detection limit of 2.0 nM. Moreover, the glutathione reductase (GR) activity can be determined by the initial rate of GSH production, i.e., the maximum PL increasing rate, with a linear range of 0.34-17.0 U/L (1 U means reduction of 1.0 µmol of oxidized glutathione per min at pH 7.6 at 25 °C) and a limit of detection of 0.34 U/L. This method allows the accurate assays of GR in clinical serum samples as well as the rapid screening of GR inhibitors, indicating its promising biomedical applications.
Assuntos
Citidina/química , Inibidores Enzimáticos/análise , Glutationa Redutase/análise , Ouro/química , Luminescência , Nanopartículas Metálicas/química , Compostos de Sulfidrila/química , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glutationa Redutase/antagonistas & inibidores , Glutationa Redutase/metabolismo , Tamanho da PartículaRESUMO
Luminol electrochemiluminescence (ECL) imaging was developed for the parallel measurement of active membrane cholesterol at single living cells, thus establishing a novel electrochemical detection technique for single cells with high analysis throughput and low detection limit. In our strategy, the luminescence generated from luminol and hydrogen peroxide upon the potential was recorded in one image so that hydrogen peroxide at the surface of multiple cells could be simultaneously analyzed. Compared with the classic microelectrode array for the parallel single-cell analysis, the plat electrode only was needed in our ECL imaging, avoiding the complexity of electrode fabrication. The optimized ECL imaging system showed that hydrogen peroxide as low as 10 µM was visible and the efflux of hydrogen peroxide from cells could be determined. Coupled with the reaction between active membrane cholesterol and cholesterol oxidase to generate hydrogen peroxide, active membrane cholesterol at cells on the electrode was analyzed at single-cell level. The luminescence intensity was correlated with the amount of active membrane cholesterol, validating our system for single-cell cholesterol analysis. The relative high standard deviation on the luminescence suggested high cellular heterogeneities on hydrogen peroxide efflux and active membrane cholesterol, which exhibited the significance of single-cell analysis. This success in ECL imaging for single-cell analysis opens a new field in the parallel measurement of surface molecules at single cells.
Assuntos
Colesterol/análise , Técnicas Eletroquímicas , Medições Luminescentes , Análise de Célula Única/métodos , Células HeLa , Humanos , Peróxido de Hidrogênio/química , Membranas/químicaRESUMO
The potential-resolved electrochemiluminescence (ECL) was achieved for the determination of two antigens at the cell surface through a potential scanning on the electrode. Luminol and Ru(bpy)3(2+) groups as ECL probes were linked with the antibodies to recognize the corresponding antigens on the cell surface. A self-quenching of luminescence from the luminol group under negative potential was initialized by the introduction of concentrated aqueous luminol, which offered accurate measurements of the luminescence from luminol and Ru(bpy)3(2+) groups under positive and negative potentials, respectively. Using this strategy, carcinoembryonic (CEA) and alphafetoprotein (AFP) antigens on cells as the models were quantified serially through a potential scanning. Different patterns of luminescence were observed at MCF 7 and PC 3 cells, which exhibited that the assay can characterize the cells with a difference expression of antigens. Compared with fluorescence measurement, the potential resolved ECL for the detection of two analytes was not limited by the spectrum difference of probes. The strategy involving potential-induced signals required a simplified optical setup and eventually offered an alternative imaging method for multiply antigens in immunohistochemistry.
Assuntos
Antígeno Carcinoembrionário/análise , Eletroquímica/métodos , Medições Luminescentes/métodos , alfa-Fetoproteínas/análise , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Eletroquímica/instrumentação , Eletrodos , Humanos , Medições Luminescentes/instrumentação , Luminol/química , Células MCF-7 , Sondas Moleculares , Compostos Organometálicos/química , Reprodutibilidade dos Testes , Dióxido de Silício , Estreptavidina/químicaRESUMO
Previously, our group has utilized the luminol electrochemiluminescence to analyze the active cholesterol at the plasma membrane in single cells by the exposure of one cell to a photomultiplier tube (PMT) through a pinhole. In this paper, fast analysis of active cholesterol at the plasma membrane in single cells was achieved by a multimicroelectrode array without the pinhole. Single cells were directly located on the microelectrodes using cell-sized microwell traps. A cycle of voltage was applied on the microelectrodes sequentially to induce a peak of luminescence from each microelectrode for the serial measurement of active membrane cholesterol. A minimal time of 1.60 s was determined for the analysis of one cell. The simulation and the experimental data exhibited a semisteady-state distribution of hydrogen peroxide on the microelectrode after the reaction of cholesterol oxidase with the membrane cholesterol, which supported the relative accuracy of the serial analysis. An eight-microelectrode array was demonstrated to analyze eight single cells in 22 s serially, including the channel switching time. The results from 64 single cells either activated by low ion strength buffer or the inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT) revealed that most of the cells analyzed had the similar active membrane cholesterol, while few cells had more active cholesterol resulting in the cellular heterogeneity. The fast single-cell analysis platform developed will be potentially useful for the analysis of more molecules in single cells using proper oxidases.
Assuntos
Membrana Celular/química , Colesterol/análise , Análise de Célula Única/métodos , Animais , Linhagem Celular , Camundongos , Microeletrodos , Análise de Célula Única/instrumentação , Fatores de TempoRESUMO
A microelectrode array has been applied for single cell analysis with relatively high throughput; however, the cells were typically cultured on the microelectrodes under cell-size microwell traps leading to the difficulty in the functionalization of an electrode surface for higher detection sensitivity. Here, nanoring electrodes embedded under the microwell traps were fabricated to achieve the isolation of the electrode surface and the cell support, and thus, the electrode surface can be modified to obtain enhanced electrochemical sensitivity for single cell analysis. Moreover, the nanometer-sized electrode permitted a faster diffusion of analyte to the surface for additional improvement in the sensitivity, which was evidenced by the electrochemical characterization and the simulation. To demonstrate the concept of the functionalized nanoring electrode for single cell analysis, the electrode surface was deposited with prussian blue to detect intracellular hydrogen peroxide at a single cell. Hundreds of picoamperes were observed on our functionalized nanoring electrode exhibiting the enhanced electrochemical sensitivity. The success in the achievement of a functionalized nanoring electrode will benefit the development of high throughput single cell electrochemical analysis.
Assuntos
Citosol/química , Técnicas Eletroquímicas , Nanoestruturas/química , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Animais , Células Cultivadas , Eletrodos , CamundongosRESUMO
A luminol electrochemiluminescence assay was reported to analyze active cholesterol at the plasma membrane in single mammalian cells. The cellular membrane cholesterol was activated by the exposure of the cells to low ionic strength buffer or the inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT). The active membrane cholesterol was reacted with cholesterol oxidase in the solution to generate a peak concentration of hydrogen peroxide on the electrode surface, which induced a measurable luminol electrochemiluminescence. Further treatment of the active cells with mevastatin decreased the active membrane cholesterol resulting in a drop in luminance. No change in the intracellular calcium was observed in the presence of luminol and voltage, which indicated that our analysis process might not interrupt the intracellular cholesterol trafficking. Single cell analysis was performed by placing a pinhole below the electrode so that only one cell was exposed to the photomultiplier tube (PMT). Twelve single cells were analyzed individually, and a large deviation on luminance ratio observed exhibited the cell heterogeneity on the active membrane cholesterol. The smaller deviation on ACAT/HMGCoA inhibited cells than ACAT inhibited cells suggested different inhibition efficiency for sandoz 58035 and mevastatin. The new information obtained from single cell analysis might provide a new insight on the study of intracellular cholesterol trafficking.
Assuntos
Membrana Celular/metabolismo , Colesterol/análise , Macrófagos/metabolismo , Análise de Célula Única/métodos , Acil Coenzima A/antagonistas & inibidores , Amidas/farmacologia , Animais , Anticolesterolemiantes/farmacologia , Transporte Biológico , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Colesterol/metabolismo , Colesterol Oxidase/química , Técnicas Eletroquímicas , Eletrodos , Peróxido de Hidrogênio/química , Lovastatina/análogos & derivados , Lovastatina/farmacologia , Medições Luminescentes , Luminol/química , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Compostos de Organossilício/farmacologia , Esterol O-Aciltransferase/antagonistas & inibidoresRESUMO
Electrochemiluminescence (ECL) is an optical readout technique that is successfully applied for the detection of biomarkers in body fluids using microbead-based immunoassays. This technology is of utmost importance for in vitro diagnostics and thus a very active research area but is mainly focused on the quest for new dyes and coreactants, whereas the investigation of the ECL optics is extremely scarce. Herein, we report the 3D imaging of the ECL signals recorded at single microbeads decorated with the ECL labels in the sandwich immunoassay format. We show that the optical effects due to the light propagation through the bead determine mainly the spatial distribution of the recorded ECL signals. Indeed, the optical simulations based on the discrete dipole approximation compute rigorously the electromagnetic scattering of the ECL emission by the microbead and allow for reconstructing the spatial map of ECL emission. Thus, it provides a global description of the ECL chemical reactivity and the associated optics. The outcomes of this 3D imaging approach complemented by the optical modeling provide insight into the ECL optics and the unique ECL chemical mechanism operating on bead-based immunoassays. Therefore, it opens new directions for mechanistic investigations, ultrasensitive ECL bioassays, and imaging.
Assuntos
Técnicas Eletroquímicas , Medições Luminescentes , Medições Luminescentes/métodos , Técnicas Eletroquímicas/métodos , Fotometria , Corantes , Imunoensaio/métodosRESUMO
Bead-based assays are successfully combined with electrochemiluminescence (ECL) technology for detection of a wide range of biomarkers. Herein, we demonstrate a novel approach to enhance the ECL signal by decorating micrometric beads with [Ru(bpy)3]2+-grafted microgels (diameter â¼100 nm). Rapid and stable light emission was spatially resolved at the level of single functionalized beads. An enhancement of the ECL signal of microgel-labeled beads by 9-fold was observed in comparison to molecularly linked [Ru(bpy)3]2+ beads prepared by a sandwich immunoassay or an amide bond. Imaging the ECL signal at the single bead level shows that the size of the ECL-emitting layer is extended using the microgels. The reported method offers a great promise for the optimization of bead-based ECL detection and subsequent development of ECL microscopy.
Assuntos
Técnicas Biossensoriais , Microgéis , Amidas , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Medições Luminescentes/métodosRESUMO
The generation of free radicals is a key process in the formation and the collapse of the bubbles in water, however, the direct and dynamic observation of the radicals in this process at single bubbles has never been achieved. Here, the hydroxyl (OH. ) and oxygen (O2 .- ) radicals at single oxygen bubbles are continuously traced using chemiluminescence (CL), in which these radicals at the bubble react with the surrounding luminol in the solution emitting the light. Varied increase trends of luminescence are observed in the generation of a bubble, floating, short parking at the water/air interface and the final explosion, revealing the complexity in the distribution of radicals at the bubble unprecedentedly. Despite more radicals are observed at the bubble generated at a deep position under the water for the stabilization, almost the same amount of radicals are included in the bubbles that is independent on the water pressure during the production of the bubble. This rich information collected from the dynamic study of bubbles illustrates the complicated generation and distribution process of radicals at the bubbles, and will facilitate the understanding of the function about the bubbles.
RESUMO
BACKGROUND: Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis. METHODS: Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age. RESULTS: Membrane cholesterol measurements are elevated in both R117H and DeltaF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTRinh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility. CONCLUSIONS: The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.
Assuntos
Membrana Celular/metabolismo , Colesterol/biossíntese , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Animais , Benzoatos/farmacologia , Sítios de Ligação , Linhagem Celular , Colesterol Oxidase/metabolismo , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Modelos Animais de Doenças , Técnicas Eletroquímicas/instrumentação , Células Epiteliais/efeitos dos fármacos , Genótipo , Humanos , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Cinética , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos CFTR , Microeletrodos , Mutação , Mucosa Nasal/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Mucosa Respiratória/metabolismo , Resveratrol , Estilbenos/farmacologia , Tiazolidinas/farmacologia , TransfecçãoRESUMO
Electrochemiluminescence (ECL) microscopy is an emerging technique with a wide range of imaging applications and unique properties in terms of high spatial resolution, surface confinement and favourable signal-to-noise ratio. Despite its successful analytical applications, tuning the depth of field (i.e., thickness of the ECL-emitting layer) is a crucial issue. Indeed, the control of the thickness of this ECL region, which can be considered as an "evanescent" reaction layer, limits the development of cell microscopy as well as bioassays. Here we report an original strategy based on chemical lens effects to tune the ECL-emitting layer in the model [Ru(bpy)3]2+/tri-n-propylamine (TPrA) system. It consists of microbeads decorated with [Ru(bpy)3]2+ labels, classically used in bioassays, and TPrA as the sacrificial coreactant. In particular we exploit the buffer capacity of the solution to modify the rate of the reactions involved in the ECL generation. For the first time, a precise control of the ECL light distribution is demonstrated by mapping the luminescence reactivity at the level of single micrometric bead. The resulting ECL image is the luminescent signature of the concentration profiles of diffusing TPrA radicals, which define the ECL layer. Therefore, our findings provide insights into the ECL mechanism and open new avenues for ECL microscopy and bioassays. Indeed, the reported approach based on a chemical lens controls the spatial extension of the "evanescent" ECL-emitting layer and is conceptually similar to evanescent wave microscopy. Thus, it should allow the exploration and imaging of different heights in substrates or in cells.
RESUMO
Electrochemiluminescence (ECL) is a powerful (bio)analytical method based on an optical readout. It is successfully applied in the heterogeneous format for immunoassays and imaging using the model and most widely used ECL system, which consists of the immobilized [Ru(bpy)3]2+ label with tripropylamine (TPA) as a coreactant. However, a major drawback is the significant decrease of the ECL intensity over time. Herein, to decipher the process responsible for this progressive loss of ECL signal, we investigated its electrochemical and photophysical properties by mapping the luminescence reactivity at the level of single micrometric beads. Polystyrene beads were functionalized by the [Ru(bpy)3]2+ dye via a sandwich immunoassay or a peptide bond. ECL emission was generated in presence of the very efficient TPA coreactant. Imaging both photoluminescence and ECL reactivities of different regions (located near or far from the electrode surface) of a [Ru(bpy)3]2+-decorated bead allows us to demonstrate the remarkable photophysical stability of the ECL label, even in presence of the very reactive electrogenerated TPA radicals. We show that the ECL vanishing correlates directly with the lower TPA oxidation current. Finally, we propose a simple electrochemical treatment, which allows to regenerate the electrode surface and thus to recover several times the strong initial ECL signal. The reactivity imaging approach provides insights into the ECL mechanism and the main factors governing the stability of the emission, which should find promising ECL applications in bioassays and microscopy.