Encapsulation in Alginates Hydrogels and Controlled Release: An Overview.

This review aims to gather the current state of the art on the encapsulation methods using alginate as the main polymeric material in order to produce hydrogels ranging from the microscopic to macroscopic sizes. The use of alginates as an encapsulation material is of growing interest, as it is fully bio-based, bio-compatible and bio-degradable. The field of application of alginate encapsulation is also extremely broad, and there is no doubt it will become even broader in the near future considering the societal demand for sustainable materials in technological applications. In this review, alginate's main properties and gelification mechanisms, as well as some factors influencing this mechanism, such as the nature of the reticulation cations, are first investigated. Then, the capacity of alginate gels to release matter in a controlled way, from small molecules to micrometric compounds, is reported and discussed. The existing techniques used to produce alginates beads, from the laboratory scale to the industrial one, are further described, with a consideration of the pros and cons with each techniques. Finally, two examples of applications of alginate materials are highlighted as representative case studies.

Ultrasensitive Imaging of Cells and Sub-Cellular Entities by Electrochemiluminescence.

Here we report on a label-free electrochemiluminescence (ECL) microscopy using exceptionally low concentrations of the [Ru(bpy)3 ]2+ luminophore. This work addresses the central point of the minimal concentration of the ECL luminophore required to image single entities. We demonstrate the possibility to record ECL images of cells and mitochondria at concentrations down to nM and pM. This is 7 orders of magnitude lower than classically-used concentrations and corresponds to a few hundreds of luminophores diffusing around the biological entities. Yet, it produces remarkably sharp negative optical contrast ECL images, as demonstrated by structural similarity index metric analyses and supported by predictions of the ECL image covering time. Finally, we show that the reported approach is a simple, fast, and highly sensitive method, which opens new avenues for ultrasensitive ECL imaging and ECL reactivity at the single molecule level.

Shadow Electrochemiluminescence Microscopy of Single Mitochondria.

Mitochondria are the subcellular bioenergetic organelles. The analysis of their morphology and topology is essential to provide useful information on their activity and metabolism. Herein, we report a label-free shadow electrochemiluminescence (ECL) microscopy based on the spatial confinement of the ECL-emitting reactive layer to image single living mitochondria deposited on the electrode surface. The ECL mechanism of the freely-diffusing [Ru(bpy)3 ]2+ dye with the sacrificial tri-n-propylamine coreactant restrains the light-emitting region to a micrometric thickness allowing to visualize individual mitochondria with a remarkable sharp negative optical contrast. The imaging approach named "shadow ECL" (SECL) reflects the negative imprint of the local diffusional hindrance of the ECL reagents by each mitochondrion. The statistical analysis of the colocalization of the shadow ECL spots with the functional mitochondria revealed by classical fluorescent biomarkers, MitoTracker Deep Red and the endogenous intramitochondrial NADH, validates the reported methodology. The versatility and extreme sensitivity of the approach are further demonstrated by visualizing single mitochondria, which remain hardly detectable with the usual biomarkers. Finally, by alleviating problems of photobleaching and phototoxicity associated with conventional microscopy methods, SECL microscopy should find promising applications in the imaging of subcellular structures.

Chemo- and Magnetotaxis of Self-Propelled Light-Emitting Chemo-electronic Swimmers.

Miniaturized autonomous chemo-electronic swimmers, based on the coupling of spontaneous oxidation and reduction reactions at the two poles of light-emitting diodes (LEDs), are presented as chemotactic and magnetotactic devices. In homogeneous aqueous media, random motion caused by a bubble-induced propulsion mechanism is observed. However, in an inhomogeneous environment, the self-propelled devices exhibit positive chemotactic behavior, propelling themselves along a pH or ionic strength gradient (∇pH and ∇I, respectively) in order to reach a thermodynamically higher active state. In addition, the intrinsic permanent magnetic moment of the LED allows self-orientation in the terrestrial magnetic field or following other external magnetic perturbations, which enables a directional motion control coupled with light emission. The interplay between chemotaxis and magnetotaxis allows fine-tuning of the dynamic behavior of these swimmers.

Does the road go up the mountain? Fictive motion between linguistic conventions and cognitive motivations.

Fictive motion (FM) characterizes the use of dynamic expressions to describe static scenes. This phenomenon is crucial in terms of cognitive motivations for language use; several explanations have been proposed to account for it, among which mental simulation (Talmy in Toward a cognitive semantics, vol 1. MIT Press, Cambridge, 2000) and visual scanning (Matlock in Studies in linguistic motivation. Mouton de Gruyter, Berlin and New York, pp 221-248, 2004a). The aims of this paper were to test these competing explanations and identify language-specific constraints. To do this, we compared the linguistic strategies for expressing several types of static configurations in four languages, French, Italian, German and Serbian, with an experimental set-up (59 participants). The experiment yielded significant differences for motion-affordance versus no motion-affordance, for all four languages. Significant differences between languages included mean frequency of FM expressions. In order to refine the picture, and more specifically to disentangle the respective roles of language-specific conventions and language-independent (i.e. possibly cognitive) motivations, we completed our study with a corpus approach (besides the four initial languages, we added English and Polish). The corpus study showed low frequency of FM across languages, but a higher frequency and translation ratio for some FM types--among which those best accounted for by enactive perception. The importance of enactive perception could thus explain both the universality of FM and the fact that language-specific conventions appear mainly in very specific contexts--the ones furthest from enaction.

Encapsulation of Active Substances in Natural Polymer Coatings.

Already used in the food, pharmaceutical, cosmetic, and agrochemical industries, encapsulation is a strategy used to protect active ingredients from external degradation factors and to control their release kinetics. Various encapsulation techniques have been studied, both to optimise the level of protection with respect to the nature of the aggressor and to favour a release mechanism between diffusion of the active compounds and degradation of the barrier material. Biopolymers are of particular interest as wall materials because of their biocompatibility, biodegradability, and non-toxicity. By forming a stable hydrogel around the drug, they provide a 'smart' barrier whose behaviour can change in response to environmental conditions. After a comprehensive description of the concept of encapsulation and the main technologies used to achieve encapsulation, including micro- and nano-gels, the mechanisms of controlled release of active compounds are presented. A panorama of natural polymers as wall materials is then presented, highlighting the main results associated with each polymer and attempting to identify the most cost-effective and suitable methods in terms of the encapsulated drug.

Easy cleaning plus stable activation of glassy carbon electrode surface by oxygen plasma.

Glassy carbon (GC) electrodes are widely used in electroanalytical applications especially in bioelectrochemistry. Their use starts with an efficient surface cleaning and activation protocol, mostly based on surface polishing steps. We studied the use of an oxygen plasma exposure of GC electrodes to replace common polishing procedures. The cyclic voltammetry (CV) responses of ferrocyanide and ferrocene-dimethanol were used to compare brand new, surface-polished and plasma-treated GC electrodes. Plasma treatment induces CV responses with improved features, close to theoretical values, as compared to other methods. The plasma effects were quasi-stable over a week when electrodes were stored in water, this being explained by increased surface energy and hydrophilicity. Furthermore, when electroreduction of diazonium was performed on GC electrodes, the surface blockade could be removed by the plasma. Thus, a short oxygen plasma treatment is prone to replace polishing protocols, that display person-dependent efficiency, in most of the experiments with GC electrodes.

A simple model of cardiac mitochondrial respiration with experimental validation.

Cardiac mitochondria are intracellular organelles that play an important role in energy metabolism and cellular calcium regulation. In particular, they influence the excitation-contraction cycle of the heart cell. A large number of mathematical models have been proposed to better understand the mitochondrial dynamics, but they generally show a high level of complexity, and their parameters are very hard to fit to experimental data. We derived a model based on historical free energy-transduction principles, and results from the literature. We proposed simple expressions that allow to reduce the number of parameters to a minimum with respect to the mitochondrial behavior of interest for us. The resulting model has thirty-two parameters, which are reduced to twenty-three after a global sensitivity analysis of its expressions based on Sobol indices. We calibrated our model to experimental data that consists of measurements of mitochondrial respiration rates controlled by external ADP additions. A sensitivity analysis of the respiration rates showed that only seven parameters can be identified using these observations. We calibrated them using a genetic algorithm, with five experimental data sets. At last, we used the calibration results to verify the ability of the model to accurately predict the values of a sixth dataset. Results show that our model is able to reproduce both respiration rates of mitochondria and transitions between those states, with very low variability of the parameters between each experiment. The same methodology may apply to recover all the parameters of the model, if corresponding experimental data were available.

Single-Particle Tracking Method in Fluorescence Microscopy to Monitor Bioenergetic Responses of Individual Mitochondria.

The spectroscopic methods commonly used to study mitochondria bioenergetics do not show the diversity of responses within a population of mitochondria (isolated or in a cell), and/or cannot measure individual dynamics. New methodological developments are necessary in order to improve quantitative and kinetic resolutions and eventually gain further insights on individual mitochondrial responses, such as studying activities of the mitochondrial permeability transition pore (mPTP ). The work reported herein is devoted to study responses of single mitochondria within a large population after isolation from cardiomyocytes. Mitochondria were preloaded with a commonly used membrane potential sensitive dye (TMRM), they are then deposited on a plasma-treated glass coverslip and subsequently energized or inhibited by additions of usual bioenergetics effectors. Responses were analyzed by fluorescence microscopy over few thousands of mitochondria simultaneously with a single organelle resolution. We report an automatic method to analyze each image of time-lapse stacks based on the TrackMate-ImageJ plug-in and specially made Python scripts. Images are processed to eliminate defects of illumination inhomogeneity, improving by at least two orders of magnitude the signal/noise ratio. This method enables us to follow the track of each mitochondrion within the observed field and monitor its fluorescence changes, with a time resolution of 400 ms, uninterrupted over the course of the experiment. Such methodological improvement is a prerequisite to further study the role of mPTP in single mitochondria during calcium transient loading.

Microwell Array Based Opto-Electrochemical Detections Revealing Co-Adaptation of Rheological Properties and Oxygen Metabolism in Budding Yeast.

Microdevices composed of microwell arrays integrating nanoelectrodes (OptoElecWell) are developed to achieve dual high-resolution optical and electrochemical detections on single Saccharomyces cerevisiae yeast cells. Each array consists of 1.6 × 105 microwells measuring 8 µm in diameter and 5 µm height, with a platinum nanoring electrode for in situ electrochemistry, all integrated on a transparent thin wafer for further high-resolution live-cell imaging. After optimizing the filling rate, 32% of cells are effectively trapped within microwells. This allows to analyse S. cerevisiae metabolism associated with basal respiration while simultaneously measuring optically other cellular parameters. In this study, the impact of glucose concentration on respiration and intracellular rheology is focused. It is found that while the oxygen uptake rate decreases with increasing glucose concentration, diffusion of tracer nanoparticles increases. The OptoElecWell-based respiration methodology provides similar results compared to the commercial gold-standard Seahorse XF analyzer, while using 20 times fewer biological samples, paving the way to achieve single cell metabolomics. In addition, it facilitates an optical route to monitor the contents within single cells. The proposed device, in combination with the dual detection analysis, opens up new avenues for measuring cellular metabolism, and relating it to cellular physiological indicators at single cell level.

Dynamic inking of large-scale stamps for multiplexed microcontact printing and fabrication of cell microarrays.

Microcontact printing has become a versatile soft lithography technique used to produce molecular micro- and nano-patterns consisting of a large range of different biomolecules. Despite intensive research over the last decade and numerous applications in the fields of biosensors, microarrays and biomedical applications, the large-scale implementation of microcontact printing is still an issue. It is hindered by the stamp-inking step that is critical to ensure a reproducible and uniform transfer of inked molecules over large areas. This is particularly important when addressing application such as cell microarray manufacturing, which are currently used for a wide range of analytical and pharmaceutical applications. In this paper, we present a large-scale and multiplexed microcontact printing process of extracellular matrix proteins for the fabrication of cell microarrays. We have developed a microfluidic inking approach combined with a magnetic clamping technology that can be adapted to most standard substrates used in biology. We have demonstrated a significant improvement of homogeneity of printed protein patterns on surfaces larger than 1 cm2 through the control of both the flow rate and the wetting mechanism of the stamp surface during microfluidic inking. Thanks to the reproducibility and integration capabilities provided by microfluidics, we have achieved the printing of three different adhesion proteins in one-step transfer. Selective cell adhesion and cell shape adaptation on the produced patterns were observed, showing the suitability of this approach for producing on-demand large-scale cell microarrays.

Fabrication of Biomolecule Microarrays for Cell Immobilization Using Automated Microcontact Printing.

Biomolecule microarrays are generally produced by conventional microarrayer, i.e., by contact or inkjet printing. Microcontact printing represents an alternative way of deposition of biomolecules on solid supports but even if various biomolecules have been successfully microcontact printed, the production of biomolecule microarrays in routine by microcontact printing remains a challenging task and needs an effective, fast, robust, and low-cost automation process. Here, we describe the production of biomolecule microarrays composed of extracellular matrix protein for the fabrication of cell microarrays by using an automated microcontact printing device. Large scale cell microarrays can be reproducibly obtained by this method.