Bimodal Electrochemiluminescence Microscopy of Single Cells.

Electrochemiluminescence (ECL) microscopy is an emerging technique with new applications such as imaging of single entities and cells. Herein, we have developed a bimodal and bicolor approach to record both positive ECL (PECL: light-emitting object on dark background) and shadow label-free ECL (SECL: nonemissive object shadowing the background luminescence) images of single cells. This bimodal approach is the result of the simultaneous emissions of [Ru(bpy)3]2+ used to label the cellular membrane (PECL) and [Ir(sppy)3]3- dissolved in solution (SECL). By spectrally resolving the ECL emission wavelengths, we recorded the images of the same cells in both PECL and SECL modes using the [Ru(bpy)3]2+ (λmax = 620 nm) and [Ir(sppy)3]3- (λmax = 515 nm) luminescence, respectively. PECL shows the distribution of the [Ru(bpy)3]2+ labels attached to the cellular membrane, whereas SECL reflects the local diffusional hindrance of the ECL reagents by each cell. The high sensitivity and surface-confined features of the reported approach are demonstrated by imaging cell-cell contacts during the mitosis process. Furthermore, the comparison of PECL and SECL images demonstrates the differential diffusion of tri-n-propylamine and [Ir(sppy)3]3- through the permeabilized cell membranes. Consequently, this dual approach enables the imaging of the morphology of the cell adhering on the surface and can significantly contribute to multimodal ECL imaging and bioassays with different luminescent systems.

Spatially Controlled CO2 Conversion Kinetics in Natural Leaves for Motion Generation.

Living systems that can spontaneously exhibit directional motion belong to diverse classes such as bacteria, sperm and plankton. They have fascinated scientists in recent years to design completely artificial or biohybrid mobile objects. Natural ingredients, like parts of plants, have been used to elaborate miniaturized dynamic objects, which can move when they are combined with other, non-natural, building blocks. Herein, we report that the precise structural tailoring of natural plant leaves allows generating a spatially predefined and confined release of oxygen gas, due to the conversion of carbon dioxide. This constitutes the driving force for generating motion, which is solely due to the respiration of leaves by photosynthesis. The rate of gas evolution can be fine-tuned by changing the light intensity and the leaf size, allowing ultimately to control the motility of objects with dimensions ranging from the millimeter to the micrometer scale.

Electrochemiluminescence Microscopy of Cells: Essential Role of Surface Regeneration.

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.

Dynamic monitoring of a bi-enzymatic reaction at a single biomimetic giant vesicle.

Giant unilamellar vesicles were used as individual biomimetic micro-reactors wherein a model bi-enzymatic reaction involving a glucose oxidase (GOx) and horseradish peroxidase (HRP) was monitored by confocal microscopy. These giant vesicles were formed from a natural mix of phospholipids in physiological conditions of pH and osmolarity (phosphate buffer, pH 7.4, 330 mOsm). The so-called Amplex Red assay, which generates the highly fluorescent resorufin species, was performed in individual vesicles and used to report on the progress of the whole reaction. We aimed at controlling kinetically and quantitatively the different steps of the bi-enzymatic reaction in vesicles. To do so, substrates (glucose and Amplex Red) were provided in individual reactors by two ways. Electro-microinjection allowed the control of volume variations owing to a reservoir of lipids connected to the vesicle membrane. Alternatively, substrates could passively diffuse from the outer solution to the vesicle compartment. The semi-permeability feature of the phospholipid membrane was characterized for all substrates and products while we demonstrated that enzymes remain sequestrated in the vesicles after their injection. The Amplex Red assay was thus achieved in individual vesicles under steady-state conditions, and could pursue over tens of minutes. Such giant vesicles are stable, fully compatible with media used for bioanalyses and allow out-of-equilibrium reactions at time scales compatible with living reaction dynamics, making them a good choice for the development of minimal cell-like systems.

Electrochemiluminescence Loss in Photobleaching.

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.

Wireless Coupling of Conducting Polymer Actuators with Light Emission.

Combining the actuation of conducting polymers with additional functionalities is an interesting fundamental scientific challenge and increases their application potential. Herein we demonstrate the possibility of direct integration of a miniaturized light emitting diode (LED) in a polypyrrole (PPy) matrix in order to achieve simultaneous wireless actuation and light emission. A light emitting diode is used as a part of an electroactive surface on which electrochemical polymerization allows direct incorporation of the electronic device into the polymer. The resulting free-standing polymer/LED hybrid can be addressed by bipolar electrochemistry to trigger simultaneously oxidation and reduction reactions at its opposite extremities, leading to a controlled deformation and an electron flow through the integrated LED. Such a dual response in the form of actuation and light emission opens up interesting perspectives in the field of microrobotics.

Potential-Induced Fine-Tuning of the Enantioaffinity of Chiral Metal Phases.

Concepts leading to single enantiomers of chiral molecules are of crucial importance for many applications, including pharmacology and biotechnology. Recently, mesoporous metal phases encoded with chiral information have been developed. Fine-tuning of the enantioaffinity of such structures by imposing an electric potential is proposed, which can influence the electrostatic interactions between the chiral metal and the target enantiomer. This allows the binding affinity between the chiral metal and the target enantiomer to be increased, and thus, the discrimination between two enantiomers to be improved. The concept is illustrated by generating chiral encoded metals in a microfluidic channel by reduction of a platinum salt in the presence of a liquid crystal and l-tryptophan as a chiral model template. After removal of the template molecules, the modified microchannel retains a pronounced chiral character. The chiral recognition efficiency of the microchannel can be fine-tuned by applying a suitable potential to the metal phase. This enables the separation of both components of a racemate flowing through the channel. The approach constitutes a promising and complementary strategy in the frame of chiral discrimination technologies.

Wireless Electromechanical Readout of Chemical Information.

Collecting electrochemical information concerning the presence of molecules in a solution is usually achieved by measuring current, potential, resistance, or impedance via connection to a power supply. Here, we suggest wireless electromechanical actuation as a straightforward readout of chemical information. This can be achieved based on the concept of bipolar electrochemistry, which allows measuring the presence of different model species in a quantitative way. We validate the concept by using a free-standing polypyrrole film. Its positively polarized extremity participates in an oxidation of the analyte and delivers electrons to the opposite extremity for the reduction of the polymer. This reduction is accompanied by the insertion of counterions and thus leads to partial swelling of the film, inducing its bending. The resulting actuation is found to be a linear function of the analyte concentration, and also a Michaelis-Menten type correlation is obtained for biochemical analytes. This electromechanical transduction allows an easy optical readout and opens up very interesting perspectives not only in the field of sensing but also far beyond, such as for the elaboration of self-regulating biomimetic systems.

Surface-Confined Electrochemiluminescence Microscopy of Cell Membranes.

Herein is reported a surface-confined microscopy based on electrochemiluminescence (ECL) that allows to image the plasma membrane of single cells at the interface with an electrode. By analyzing photoluminescence (PL), ECL and AFM images of mammalian CHO cells, we demonstrate that, in contrast to the wide-field fluorescence, ECL emission is confined to the immediate vicinity of the electrode surface and only the basal membrane of the cell becomes luminescent. The resulting ECL microscopy reveals details that are not resolved by classic fluorescence microscopy, without any light irradiation and specific setup. The thickness of the ECL-emitting regions is ∼500 nm due to the unique ECL mechanism that involves short-lifetime electrogenerated radicals. In addition, the reported ECL microscopy is a dynamic technique that reflects the transport properties through the cell membranes and not only the specific labeling of the membranes. Finally, disposable transparent carbon nanotube (CNT)-based electrodes inkjet-printed on classic microscope glass coverslips were used to image cells in both reflection and transmission configurations. Therefore, our approach opens new avenues for ECL as a surface-confined microscopy to develop single cell assays and to image the dynamics of biological entities in cells or in membranes.

Preparation of Template-Free Robust Yolk-Shell Gelled Particles from Controllably Evolved All-in-Water Emulsions.

A template-free all-aqueous bulk preparation of robust hollow capsules having a gelatin shell from all-in-water double emulsions is reported. The hot (>40 °C) quaternary system water/polyethylene glycol (PEG)/gelatin/alginate is shown to spontaneously form PEG-in-gelatin-in-PEG double water emulsion droplets having a multinuclear core. These droplets are stable upon cooling below the temperature at which gelatin gelled. In contrast, above the melting temperature of gelatin, multinuclear double emulsion droplets controllably evolve into stable mononuclear yolk (aqueous PEG)-shell (gelatin) capsules dispersed in the aqueous PEG continuous phase. It is demonstrated that the gelatin shell can accommodate negatively charged latex beads and be re-enforced by glutaraldehyde or silica. These capsules are also shown to encapsulate payloads, suggesting possible applications in microencapsulation, drug delivery, and synthetic biology.

Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels.

Multiplexed bioluminescence resonance energy transfer (BRET) assays were developed to monitor the activation of several functional transient receptor potential (TRP) channels in live cells and in real time. We probed both TRPV1 intramolecular rearrangements and its interaction with Calmodulin (CaM) under activation by chemical agonists and temperature. Our BRET study also confirmed that: (1) capsaicin and heat promoted distinct transitions, independently coupled to channel gating, and that (2) TRPV1 and Ca2+-bound CaM but not Ca2+-free CaM were preassociated in resting live cells, while capsaicin activation induced both the formation of more TRPV1/CaM complexes and conformational changes. The BRET assay, based on the interaction with Calmodulin, was successfully extended to TRPV3 and TRPV4 channels. We therefore developed a full-spectral three-color BRET assay for analyzing the specific activation of each of the three TRPV channels in a single sample. Such key improvement in BRET measurement paves the way for the simultaneous monitoring of independent biological pathways in live cells.

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.

Wireless Electrochemical Actuation of Conducting Polymers.

Electrochemical actuation of conducting polymers usually requires a direct connection to an electric power supply. In this contribution, we suggest to overcome this issue by using the concept of bipolar electrochemistry. This allows changing the oxidation state of the polymer in a gradual and wireless way. Free-standing polypyrrole films were synthesized with an intrinsic morphological asymmetry of their two faces in order to form a bilayer structure. Immersing such objects in an electrolyte solution and exposing them to a potential gradient leads to the asymmetric oxidation/reduction of the polymer, resulting in differential shrinking and swelling along the main axis. This additional asymmetry is responsible for a structural deformation. Optimization allowed highly efficient bending, which is expected to open up completely new directions in the field of actuation due to the wireless mode of action.

Coupling Electrochemistry with Fluorescence Confocal Microscopy To Investigate Electrochemical Reactivity: A Case Study with the Resazurin-Resorufin Fluorogenic Couple.

The redox couple resazurin-resorufin exhibits electrofluorochromic properties which are investigated herein by absorption and fluorescence spectroelectrochemistry and by electrochemically coupled-fluorescence confocal laser scanning microscopy (EC-CLSM). At pH 10, the highly fluorescent resorufin dye is generated at the electrode surface by the electrochemical reduction of the poorly fluorescent resazurin. Performing EC-CLSM at electrode surfaces allows to monitor spatially resolved electrochemical processes in situ and in real time. Using a small (315 µm diameter) cylindrical electrode, a steady-state diffusion layer builds up under potentiostatic conditions at -0.45 V vs Ag|AgCl. Mapping the fluorescence intensity in 3D by CLSM enables us to reconstruct the relative concentration profile of resorufin around the electrode. The comparison of the experimental diffusion-profile with theoretical predictions demonstrates that spontaneous convection has a direct influence on the actual thickness of the diffusion layer, which is smaller than the value predicted for a purely diffusional transport. This study shows that combining fluorescence CLSM with electrochemistry is a powerful tool to study electrochemical reactivity at a spatially resolved level.

Direct oxidative pathway from amplex red to resorufin revealed by in situ confocal imaging.

Amplex Red (AR) is a very useful chemical probe that is employed in biochemical assays. In these assays, the non-fluorescent AR is converted to resorufin (RS), which strongly absorbs in the visible region (λabs = 572 nm) and yields strong fluorescence (λfluo = 583 nm). Even if AR is commonly used to report on enzymatic oxidase activities, an increasing number of possible interferences have been reported, thus lowering the accuracy of the so-called AR assay. As a redox-based reaction, we propose here to directly promote the conversion of AR to RS by means of electrochemistry. The process was first assessed by classic electrochemical and spectroelectrochemical investigations. In addition, we imaged the electrochemical conversion of AR to RS at the electrode surface by in situ confocal microscopy. The coupling of methodologies allowed to demonstrate that RS is directly formed from AR by an oxidation step, unlike what was previously reported. This gives a new insight in the deciphering of AR assays' mechanism and about their observed discrepancy.

Effects of 50 Hz magnetic fields on gap junctional intercellular communication in NIH3T3 cells.

The present study focused on gap junctional intercellular communication (GJIC) as a target for biological effects of extremely low-frequency (ELF) magnetic field (MF) exposure. Fluorescence recovery after photobleaching microscopy (FRAP) was used to visualize diffusion of a fluorescent dye between NIH3T3 fibroblasts through gap junctions. The direct effect of 24 h exposure to 50 Hz MF at 0.4 or 1 mT on GJIC function was assessed in one series of experiments. The potential synergism of MF with an inhibitor of GJIC, phorbol ester (TPA), was studied in another series by observing FRAP when NIH3T3 cells were incubated with TPA for 1 h following 24 h exposure to MF. In contrast to other reports of ELF-MF effects on GJIC, under our experimental conditions we observed neither direct inhibition of GJIC nor synergism with TPA-induced inhibition from 50 Hz MF exposures.

Imaging redox activity at bipolar electrodes by indirect fluorescence modulation.

Bipolar electrochemistry (BPE) is nowadays well-known but relatively underexploited and still considered as unconventional. It has been used, among others, in the frame of materials science and most importantly has also found very promising applications in analytical chemistry. Here, we extend this emerging field of analytical applications to the development of a new sensing concept based on indirect BPE. This approach is based on the generation of local pH gradients which will allow detecting indirectly redox-active molecules due to a modulation of the fluorescence intensity in the vicinity of a bipolar electrode.

Optical microwell array for large scale studies of single mitochondria metabolic responses.

Microsystems based on microwell arrays have been widely used for studies on single living cells. In this work, we focused on the subcellular level in order to monitor biological responses directly on individual organelles. Consequently, we developed microwell arrays for the entrapment and fluorescence microscopy of single isolated organelles, mitochondria herein. Highly dense arrays of 3-µm mean diameter wells were obtained by wet chemical etching of optical fiber bundles. Favorable conditions for the stable entrapment of individual mitochondria within a majority of microwells were found. Owing to NADH auto-fluorescence, the metabolic status of each mitochondrion was analyzed at resting state (Stage 1), then following the addition of a respiratory substrate (Stage 2), ethanol herein, and of a respiratory inhibitor (Stage 3), antimycin A. Mean levels of mitochondrial NADH were increased by 29% and 35% under Stages 2 and 3, respectively. We showed that mitochondrial ability to generate higher levels of NADH (i.e., its metabolic performance) is not correlated either to the initial energetic state or to the respective size of each mitochondrion. This study demonstrates that microwell arrays allow metabolic studies on populations of isolated mitochondria with a single organelle resolution.

Infrared photoinduced electrochemiluminescence microscopy of single cells.

Electrochemiluminescence (ECL) is evolving rapidly from a purely analytical technique into a powerful microscopy. Herein, we report the imaging of single cells by photoinduced ECL (PECL; λem = 620 nm) stimulated by an incident near-infrared light (λexc = 1050 nm). The cells were grown on a metal-insulator-semiconductor (MIS) n-Si/SiOx/Ir photoanode that exhibited stable and bright PECL emission. The large anti-Stokes shift allowed for the recording of well-resolved images of cells with high sensitivity. PECL microscopy is demonstrated at a remarkably low onset potential of 0.8 V; this contrasts with classic ECL, which is blind at this potential. Two imaging modes are reported: (i) photoinduced positive ECL (PECL+), showing the cell membranes labeled with the [Ru(bpy)3]2+ complex; and (ii) photoinduced shadow label-free ECL (PECL-) of cell morphology, with the luminophore in the solution. Finally, by adding a new dimension with the near-infrared light stimulus, PECL microscopy should find promising applications to image and study single photoactive nanoparticles and biological entities.

Monitoring metabolic responses of single mitochondria within poly(dimethylsiloxane) wells: study of their endogenous reduced nicotinamide adenine dinucleotide evolution.

It is now demonstrated that mitochondria individually function differently because of specific energetic needs in cell compartments but also because of the genetic heterogeneity within the mitochondrial pool-network of a cell. Consequently, understanding mitochondrial functioning at the single organelle level is of high interest for biomedical research, therefore being a target for analyticians. In this context, we developed easy-to-build platforms of milli- to microwells for fluorescence microscopy of single isolated mitochondria. Poly(dimethylsiloxane) (PDMS) was determined to be an excellent material for mitochondrial deposition and observation of their NADH content. Because of NADH autofluorescence, the metabolic status of each mitochondrion was analyzed following addition of a respiratory substrate (stage 2), ethanol herein, and a respiratory inhibitor (stage 3), Antimycin A. Mean levels of mitochondrial NADH were increased by 32% and 62% under stages 2 and 3, respectively. Statistical studies of NADH value distributions evidenced different types of responses, at least three, to ethanol and Antimycin A within the mitochondrial population. In addition, we showed that mitochondrial ability to generate high levels of NADH, that is its metabolic performance, is not correlated either to the initial energetic state or to the respective size of each mitochondrion.