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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; λ em = 620 nm) stimulated by an incident near-infrared light ( λ exc = 1050 nm). The cells were grown on a metal-insulator-semiconductor (MIS) n-Si/SiO x /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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Localized photoinduced electrochemiluminescence (PECL) is studied on photoanodes composed of Ir microbands deposited on n-Si/SiO x . We demonstrate that PECL microscopy precisely imaged the hole-driven heterogeneous photoelectrochemical reactivity. The method is promising for elucidating the local activity of photoelectrodes that are employed in solar energy conversion.

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+ (λ max = 620 nm) and [Ir(sppy) 3 ] 3- (λ max = 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.

Hypothesis: Colloidosomes made of stimuli-responsive microgels offer the opportunity to design polymeric capsules with a hierarchical and tunable pore distribution. Coacervates stabilized by a microgel monolayer represent a unique strategy to build colloidosomes from all-aqueous emulsion drops, while exploiting the sequestration and dissolution properties of the coacervates., Experiments: Methacrylated poly(N-isopropylacrylamide) (pNIPAM) microgels are used to stabilize coacervates made of an ampholyte polymer at a pH close to its isoelectric point. They are further cross-linked under UV-irradiation. The resulting assemblies are studied by means of confocal microscopy. Their permeability towards dextrans and nanoparticles is studied before and after dissolution of the coacervate., Findings: PNIPAM microgels are found to stabilize the coacervates by adsorbing at their surface. Inter cross-linking the microgels results in the formation of an elastic colloidosome that persists after the coacervate dissolution and withstands surface deformations up to about 200%. The coacervate is exploited as a sequestrating core to entrap a water-soluble payload, which can be further released upon coacervate dissolution, while the membrane exhibits a size-selecting permeability. The membrane properties can also be switched by the volume phase transition of the microgels. Coacervate-embedded colloidosomes open new perspectives in the area of encapsulation/extraction and controlled transport of water-soluble/dispersed species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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 ∼100 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Chiral-imprinted mesoporous Pt-Ir alloy surfaces were combined in a synergetic way with electrochemiluminescence (ECL) to detect the two enantiomers of phenylalanine (PA) as a model compound, acting simultaneously as a chiral target and as a co-reactant to generate significant differences in ECL signals. The chiral features of the metal surfaces are converted into an enantioselective electrogeneration of the excited state of the [Ru(bpy) 3 ] 2+ dye, which in fine produces the differentiating light emission with up to 20-fold differences in intensity for the two enantiomers. These findings open up the possibility of developing new ECL-based bioassays and microscopy of chiral environments.

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., (© 2022 Wiley-VCH GmbH.)

In situ UV-vis-NIR spectroelectrochemistry has been intensively used to evaluate the electronic transitions during the charging/discharging process of π-conjugated polymers. However, the type of charge carrier and the mechanisms of their transport, remains still a point of discussion. Herein, the coupling between UV-vis-NIR spectroscopy and in situ electrochemical-conductance measurements is proposed to compare the doping process of three different thiophene-based conducting polymers. The simultaneous monitoring of electrical and absorption properties, associated with low energy electronic transitions characteristic for polarons and bipolarons, was achieved. In addition, this method allows evaluating the reversible charge trapping mechanism of poly-3,4- o -xylendioxythiophene (PXDOT), caused by the formation of σ-dimers, making it a very useful tool to determine relevant physicochemical properties of conductive materials.

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., (© 2021 Wiley-VCH GmbH.)

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.

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.

Rational design and shaping of soft smart materials offer potential applications that cannot be addressed with rigid systems. In particular, electroresponsive elastic materials are well-suited for developing original active devices, such as pumps and actuators. However, applying the electric stimulus requires usually a physical connection between the active part and a power supply. Here we report about the design of an electromechanical system based on conducting polymers, enabling the actuation of a wireless microfluidic pump. Using the electric field-induced asymmetric polarization of miniaturized polypyrrole tubes, it is possible to trigger simultaneously site-specific chemical reactions, leading to shrinking and swelling in aqueous solution without any physical connection to a power source. The complementary electrochemical reactions occurring at the opposite extremities of the tube result in a differential change of its diameter. In turn, this electromechanical deformation allows inducing highly controlled fluid dynamics. The performance of such a remotely triggered electrochemically active soft pump can be fine-tuned by optimizing the wall thickness, length and inner diameter of the material. The efficient and fast actuation of the polymer pump opens up new opportunities for actuators in the field of fluidic or microfluidic devices, such as controlled drug release, artificial organs and bioinspired actuators., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this work, bipolar electrochemistry is used to perform wireless indirect electrodeposition of two different polymer coatings on both sides of carbon nanotube arrays. Using a thermoresponsive hydrogel on one side and an inert insoluble polymer on the other side, it is possible to generate, in a single step, a nanoporous reservoir with Janus character closed on one side by a thermoresponsive membrane. The thermoresponsive polymer, poly( N -isopropylacrylamide) (pNIPAM), is generated by the local reduction of persulfate ions, which initiates radical polymerization of NIPAM. Electrophoretic paint (EP) is chosen as an inert polymer. It is deposited by precipitation because of a local decrease in pH during water oxidation. Both polymers can be deposited simultaneously on opposite sides of the bipolar electrode during the application of the electric field, yielding a double-modified Janus object. Moreover, the length and thickness of the polymer layers can be controlled by varying the electric field and the deposition time. This concept is applied to vertically aligned carbon nanotube arrays (VACNTs), trapped inside an anodic aluminum oxide membrane, which can further be used as a smart reservoir for chemical storage and release. A fluorescent dye is loaded in the VACNTs and its release is studied as a function of temperature. Low temperature, when the hydrogel layer is in the swollen state, allows diffusion of the molecule. Dye release occurs on the hydrogel-modified side of the VACNTs. At high temperatures, when the hydrogel layer is in the collapsed state, dye release is blocked because of the impermeability of the pNIPAM layer. This concept paves the way toward the design of advanced devices in the fields of drug storage and directed delivery.

We report the synthesis of a hybrid bilayer, being composed of a free-standing conducting polymer film and a layer of mesoporous metal, encoded with chiral features. The resulting structure constitutes an enantioselective actuator, which can be electrochemically addressed in a wireless way. The controlled discriminatory deformation of the film allows an easy readout of chiral information.

Coacervation is a phase separation process involving two aqueous phases, one solute-phase and one solute-poor phase. It is frequently observed among oppositely-charged polyelectrolyte systems. In this study, we focus on self-coacervation involving a single polymer chain and investigate its potential for encapsulation applications. Negatively charged polyacrylic acid polymer chains were partially cationized using diamine and carbodiimide chemistry affording ampholytes, named PAA-DA, with tunable charge ratio. When dispersed in water, at pH 7, PAA-DA was soluble but a phase separation occurs when decreasing pH close to the isoelectric point. Coacervation is found only for a given amine-to-acid ratio otherwise precipitation is observed. Increasing the pH above 4 yielded progressive destruction of the coacervates droplets via the formation of vacuoles within droplets and subsequent full homogeneous redispersion of PAA-DA in water. However, addition of calcium allowed increasing the coacervate droplet stability upon increasing the pH to 7 as the divalent ion induced gelation within droplets. Moreover, the coacervate droplets present the ability to spontaneously sequestrate a broad panel of entities, from small molecules to macromolecules or colloids, with different charges, size and hydrophobicity. Thanks to the reversible character of the coacervates, triggered-release could be easily achieved, either by varying the pH or by removing calcium ions in the case of calcium-stabilized coacervates. Self-coacervation presents the advantage of pathway-independent preparation, offering a real output interest in pharmacy, water treatment, food science or diagnostics., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A simple route to deliver on demand hydrosoluble molecules such as peptides, packaged in biocompatible and biodegradable microgels, is presented. Hyaluronic acid hydrogel particles with a controlled structure are prepared using a microfluidic approach. Their porosity and their rigidity can be tuned by changing the crosslinking density. These negatively-charged polyelectrolytes interact strongly with positively-charged linear peptides such as poly-l-lysine (PLL). Their interactions induce microgel deswelling and inhibit microgel enzymatic degradability by hyaluronidase. While small PLL penetrate the whole volume of the microgel, PLL larger than the mesh size of the network remain confined at its periphery. They make a complexed layer with reduced pore size, which insulates the microgel inner core from the outer medium. Consequently, enzymatic degradation of the matrix is fully inhibited and non-affinity hydrophilic species can be trapped in the core. Indeed, negatively-charged or small neutral peptides, without interactions with the network, usually diffuse freely across the network. By simple addition of large PLL, they are packaged in the core and can be released on demand, upon introduction of an enzyme that degrades selectively the capping agent. Single polyelectrolyte layer appears as a simple generic method to coat hydrogel-based materials of various scales for encapsulation and controlled delivery of hydrosoluble molecules., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

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.

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., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Phospholipid Giant Unilamellar Vesicles (GUVs) are usually prepared by electroformation in water, that is in a low-conductivity solution. We developed a protocol allowing their electroformation in the most common physiological buffer, phosphate-buffered saline (PBS). This was achieved based on a specific sequence of increasing electrical fields and for the two usual electrode types for electroformation, namely Indium Tin oxide-coated glass slides and Pt electrodes. These GUVs are stable over time (hour time-scale) and they can be isolated or micro-injected. The membrane composition was modified by adding cholesterol in order to adjust its mechanical properties. The optimal proportion of cholesterol vs. total phospholipid concentration was a ratio of 20 mol%, which increases membrane rigidity and facilitates vesicle microinjection.

The coupling between electrochemistry and fluorescence confocal laser scanning microscopy (FCLSM) allows deciphering the electrochemical and/or redox reactivity of electroactive fluorophores. This is demonstrated with phenoxazine electrofluorogenic species frequently used in bioassays by mapping the variation of fluorescence intensity with respect to the distance from the electrode. The electrochemical conversion of resorufin dye (RF) to non-fluorescent dihydroresorufin (DH) leads to a sharp decrease of the fluorescence signal in the vicinity of the electrode. In contrast, the direct reduction of resazurin (RZ) to DH leads to an unexpected maximum fluorescence intensity localized further away from the surface. This observation indicates that the initial electron transfer (heterogeneous) is followed by a chemical comproportionation step (homogeneous), leading to the formation of RF within the diffusion layer with a characteristic concentration profile. Therefore, in situ FCLSM affords a direct way to monitor such chemical reactivity in space and to decipher a new redox pathway that cannot be resolved solely by electrochemical means.

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.

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., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Controlling the interface between biological tissues and electrodes remains an important challenge for the development of implantable devices in terms of electroactivity, biocompatibility, and long-term stability. To engineer such a biocompatible interface a low molecular weight gel (LMWG) based on a glycosylated nucleoside fluorocarbon amphiphile (GNF) was employed for the first time to wrap gold electrodes via a noncovalent anchoring strategy, that is, self-assembly of GNF at the electrode surface. Scanning electron microscopy (SEM) studies indicate that the gold surface is coated with the GNF hydrogels. Electrochemical measurements using cyclic voltammetry (CV) clearly show that the electrode properties are not affected by the presence of the hydrogel. This coating layer of 1 to 2 μm does not significantly slow down the mass transport through the hydrogel. Voltammetry experiments with gel coated macroporous enzyme electrodes reveal that during continuous use their current is improved by 100% compared to the noncoated electrode. This demonstrates that the supramolecular hydrogel dramatically increases the stability of the bioelectrochemical interface. Therefore, such hybrid electrodes are promising candidates that will both offer the biocompatibility and stability needed for the development of more efficient biosensors and biofuel cells.

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 Ca 2+ -bound CaM but not Ca 2+ -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., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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.

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.

Two bolaamphiphilic quadrupoles built from a fluorene core conjugated with azine endgroups were designed and successfully used to stain GUV membranes. Their quadrupolar character induces both a shift of the pKa values close to the physiological pH and large two-photon absorption responses (i.e. over 1000 GM for acidic forms). As such they hold promise as ratiometric two-photon pH probes for monitoring slight variations of pH near cell membranes using two-photon excitation in the NIR region.

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., (© 2015 Wiley Periodicals, Inc.)

Most of the methods dedicated to the monitoring of metabolic responses from isolated mitochondria are based on whole-population analyses. They rarely offer an individual resolution though fluorescence microscopy allows it, as demonstrated by numerous studies on single mitochondria activities in cells. Herein, we report on the preparation and use of microwell arrays for the entrapment and fluorescence microscopy of single isolated mitochondria. Highly dense arrays of 3 μm mean diameter wells were obtained by the chemical etching of optical fiber bundles (850 μm whole diameter). They were manipulated by a micro-positioner and placed in a chamber made of a biocompatible elastomer (polydimethylsiloxane or PDMS) and a glass coverslip, on the platform of an inverted microscope. The stable entrapment of individual mitochondria (extracted from Saccharomyces cerevisiae yeast strains, inter alia, expressing a green fluorescent protein) within the microwells was obtained by pretreating the optical bundles with an oxygen plasma and dipping the hydrophilic surface of the array in a concentrated solution of mitochondria. Based on the measurement of variations of the intrinsic NADH fluorescence of each mitochondrion in the array, their metabolic status was analyzed at different energetic respiratory stages: under resting state, following the addition of an energetic substrate to stimulate respiration (ethanol herein) and the addition of a respiratory inhibitor (antimycin A). Statistical analyses of mean variations of mitochondrial NADH in the population were subsequently achieved with a single organelle resolution.

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.

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.

The site selective electrodeposition of silver metal onto a conducting object such as carbon microtubes (CMTs) in an electrolytic solution could be achieved by means of bipolar electrochemistry. Two half reactions are simultaneously carried out at both extremities of the CMT, which act as a bipolar electrode. The thermodynamic threshold value of the process, which consists in metal electroreduction and concomitant water oxidation is directly related to the length of CMT. That is the reason why, when scaling down the methodology to microscale objects, electric fields in the range of tenths of kilovolts per meter are necessary. In that context, a CE apparatus provides a convenient experimental platform to achieve in a straightforward manner such experimental conditions. We exemplify this methodology with the efficient and quick electroreduction of Ag⁺ on CMTs from a low-concentration aqueous electrolytic solution during the migration across a fused capillary. CE allows applying safely a large enough electric field (typically ∼30 kV/m) for the successful modification of 15 to 20 μm-long substrates. The corresponding hybrid materials have been characterized by optical microscopy as well as SEM and energy-dispersive X-ray spectroscopy., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Myofibrillar myopathies (MFMs) are an expanding and increasingly recognized group of neuromuscular disorders caused by mutations in DES, CRYAB, MYOT, and ZASP. The latest gene to be associated with MFM was FLNC; a p.W2710X mutation in the 24th immunoglobulin-like repeat of filamin C was shown to be the cause of a distinct type of MFM in several German families. We studied an International cohort of 46 patients from 39 families with clinically and myopathologically confirmed MFM, in which DES, CRYAB, MYOT, and ZASP mutations have been excluded. In patients from an unrelated family a 12-nucleotide deletion (c.2997_3008del) in FLNC resulting in a predicted in-frame four-residue deletion (p.Val930_Thr933del) in the seventh repeat of filamin C was identified. Both affected family members, mother and daughter, but not unrelated control individuals, carried the p.Val930_Thr933del mutation. The mutation is transcribed and, based on myopathological features and immunoblot analysis, it leads to an accumulation of dysfunctional filamin C in the myocytes. The study results suggest that the novel p.Val930_Thr933del mutation in filamin C is the cause of MFM but also indicate that filamin C mutations are a comparatively rare cause of MFM.