Stability Study of Synthetic Diamond Using a Thermally Controlled Biological Environment: Application towards Long-Lasting Neural Prostheses.

This paper demonstrates, for the first time, the stability of synthetic diamond as a passive layer within neural implants. Leveraging the exceptional biocompatibility of intrinsic nanocrystalline diamond, a comprehensive review of material aging analysis in the context of in-vivo implants is provided. This work is based on electric impedance monitoring through the formulation of an analytical model that scrutinizes essential parameters such as the deposited metal resistivity, insulation between conductors, changes in electrode geometry, and leakage currents. The evolution of these parameters takes place over an equivalent period of approximately 10 years. The analytical model, focusing on a fractional capacitor, provides nuanced insights into the surface conductivity variation. A comparative study is performed between a classical polymer material (SU8) and synthetic diamond. Samples subjected to dynamic impedance analysis reveal distinctive patterns over time, characterized by their physical degradation. The results highlight the very high stability of diamond, suggesting promise for the electrode's enduring viability. To support this analysis, microscopic and optical measurements conclude the paper and confirm the high stability of diamond and its strong potential as a material for neural implants with long-life use.

Oxidative stress activates red cell adhesion to laminin in sickle cell disease.

Vaso-occlusive crises are the hallmark of sickle cell disease (SCD). They are believed to occur in two steps, starting with adhesion of deformable low-dense red blood cells (RBCs), or other blood cells such as neutrophils, to the wall of post-capillary venules, followed by trapping of the denser RBCs or leukocytes in the areas of adhesion because of reduced effective lumen-diameter. In SCD, RBCs are heterogeneous in terms of density, shape, deformability and surface proteins, which accounts for the differences observed in their adhesion and resistance to shear stress. Sickle RBCs exhibit abnormal adhesion to laminin mediated by Lu/BCAM protein at their surface. This adhesion is triggered by Lu/BCAM phosphorylation in reticulocytes but such phosphorylation does not occur in mature dense RBCs despite firm adhesion to laminin. In this study, we investigated the adhesive properties of sickle RBC subpopulations and addressed the molecular mechanism responsible for the increased adhesion of dense RBCs to laminin in the absence of Lu/BCAM phosphorylation. We provide evidence for the implication of oxidative stress in post-translational modifications of Lu/BCAM that impact its distribution and cis-interaction with glycophorin C at the cell surface activating its adhesive function in sickle dense RBCs.

Effects of Poloxamer 188 on red blood cell membrane properties in sickle cell anaemia.

Vaso-occlusive crisis (VOC) is the main acute complication in sickle cell anaemia (SS) and several clinical trials are investigating different drugs to improve the clinical severity of SS patients. A phase III study is currently exploring the profit of Velopoloxamer in SS during VOCs. We analysed, in-vitro, the effect of poloxamer (P188) on red blood cell (RBC) properties by investigating haemorheology, mechanical and adhesion functions using ektacytometry, microfluidics and dynamic adhesion approaches, respectively. We show that poloxamer significantly reduces blood viscosity, RBC aggregation and adhesion to endothelial cells, supporting the beneficial use of this molecule in SS therapy.

Splenic retention of Plasmodium falciparum gametocytes to block the transmission of malaria.

Plasmodium falciparum is transmitted from humans to Anopheles mosquito vectors via the sexual erythrocytic forms termed gametocytes. Erythrocyte filtration through microsphere layers (microsphiltration) had shown that circulating gametocytes are deformable. Compounds reducing gametocyte deformability would induce their splenic clearance, thus removing them from the blood circulation and blocking malaria transmission. The hand-made, single-sample prototype for microsphiltration was miniaturized to a 96-well microtiter plate format, and gametocyte retention in the microsphere filters was quantified by high-content imaging. The stiffening activity of 40 pharmacological compounds was assessed in microtiter plates, using a small molecule (calyculin) as a positive control. The stiffening activity of calyculin was assessed in spleen-mimetic microfluidic chips and in macrophage-depleted mice. Marked mechanical retention (80% to 90%) of mature gametocytes was obtained in microplates following exposure to calyculin at concentrations with no effect on parasite viability. Of the 40 compounds tested, including 20 antimalarials, only 5 endoperoxides significantly increased gametocyte retention (1.5- to 2.5-fold; 24 h of exposure at 1 µM). Mature gametocytes exposed to calyculin accumulated in microfluidic chips and were cleared from the circulation of macrophage-depleted mice as rapidly as heat-stiffened erythrocytes, thus confirming results obtained using the microsphiltration assay. An automated miniaturized approach to select compounds for their gametocyte-stiffening effect has been established. Stiffening induces gametocyte clearance both in vitro and in vivo. Based on physiologically validated tools, this screening cascade can identify novel compounds and uncover new targets to block malaria transmission. Innovative applications in hematology are also envisioned.

Monitoring the permeabilization of a single cell in a microfluidic device, through the estimation of its dielectric properties based on combined dielectrophoresis and electrorotation in situ experiments.

The electric field is commonly used in microdevices to handle, treat, or monitor living cells for various biological or biomedical applications (cells electrofusion, gene electrotransfer, drugs injection, cell sorting, …). Dielectrophoresis (DEP) forces, using stationary waves (conventional DEP) or traveling waves, are widely used for the cell handling or sorting. Electrorotation, which is induced by a rotating electrical field, is used for the determination of cell dielectric parameters. The application of pulsed electric field (PEF) results in the cell membrane permeabilization that might allow the transfer of various molecules in the cytoplasm. In this paper, we propose a method to monitor in situ the level of electropermeabilization induced by PEF application on a single cell, by combining the dielectrophoresis force and the electrorotation torque within a microfluidic device. The method was experimented on two different cell lines (human leukemic T-cell lymphoblast and murine melanoma cell): a single cell is captured by dielectrophoresis while its dielectric properties (both permittivity and conductivity of cytoplasm and membrane) are estimated thanks to a rotating electric field, which is applied simultaneously. The permeabilization effect of PEF, applied to the single cell trapped in such conditions in the biodevice, could be monitored by the estimation of its dielectric properties before and after pulse application.

A biomimetic microfluidic chip to study the circulation and mechanical retention of red blood cells in the spleen.

Red blood cells (RBCs) are deformable and flow through vessels narrower than their own size. Their deformability is most stringently challenged when they cross micrometer-wide slits in the spleen. In several inherited or acquired RBC disorders, blockade of small vessels by stiff RBCs can trigger organ damage, but a functional spleen is expected to clear these abnormal RBCs from the circulation before they induce such complications. We analyzed flow behavior of RBCs in a microfluidic chip that replicates the mechanical constraints imposed on RBCs as they cross the human spleen. Polymer microchannels obtained by soft lithography with a hydraulic diameter of 25 µm drove flow into mechanical filtering units where RBCs flew either slowly through 5- to 2-µm-wide slits or rapidly along 10-µm-wide channels, these parallel paths mimicking the splenic microcirculation. Stiff heated RBCs accumulated in narrow slits seven times more frequently than normal RBCs infused simultaneously. Stage-dependent retention of Plasmodium falciparum-infected RBCs was also observed in these slits. We also analyzed RBCs from patients with hereditary spherocytosis and observed retention for those having the most altered mechanical properties as determined by ektacytometry. Thus, in keeping with previous observations in vivo and ex vivo, the chip successfully discriminated poorly deformable RBCs based on their distinct mechanical properties and on the intensity of the cell alteration. Applications to the exploration of the pathogenesis of malaria, hereditary spherocytosis, sickle cell disease and other RBC disorders are envisioned.

Surface Acoustic Wave-Based Microfluidic Device for Microparticles Manipulation: Effects of Microchannel Elasticity on the Device Performance.

Size sorting, line focusing, and isolation of microparticles or cells are fundamental ingredients in the improvement of disease diagnostic tools adopted in biology and biomedicine. Microfluidic devices are exploited as a solution to transport and manipulate (bio)particles via a liquid flow. Use of acoustic waves traveling through the fluid provides non-contact solutions to the handling goal, by exploiting the acoustophoretic phenomenon. In this paper, a finite element model of a microfluidic surface acoustic wave-based device for the manipulation of microparticles is reported. Counter-propagating waves are designed to interfere inside a PDMS microchannel and generate a standing surface acoustic wave which is transmitted to the fluid as a standing pressure field. A model of the cross-section of the device is considered to perform a sensitivity analysis of such a standing pressure field to uncertainties related to the geometry of the microchannel, especially in terms of thickness and width of the fluid domain. To also assess the effects caused by possible secondary waves traveling in the microchannel, the PDMS is modeled as an elastic solid material. Remarkable effects and possible issues in microparticle actuation, as related to the size of the microchannel, are discussed by way of exemplary results.

Activity monitoring of functional OprM using a biomimetic microfluidic device.

This paper describes the fabrication and use of a biomimetic microfluidic device for the monitoring of a functional porin reconstituted within a miniaturized suspended artificial bilayer lipid membrane (BLM). Such a microfluidic device allows for (1) fluidic and electrical access to both sides of the BLM and (2) reproducible membrane protein insertion and long-term electrical monitoring of its conductance (G(i)), thanks to the miniaturization of the BLM. We demonstrate here for the first time the feasibility to insert a large trans-membrane protein through its ß-barrel, and monitor its functional activity for more than 1 hour (limited by buffer evaporation). In this paper, we specifically used our device for the monitoring of OprM, a bacterial efflux channel involved in the multidrug resistance of the bacteria Pseudomonas aeruginosa. Sub-steps of the OprM channel conductance were detected during the electrical recordings within our device, which might be due to oscillations between several structural conformations (sub-states) adopted by the protein, as part of its opening mechanism. This work is a first step towards the establishment of a genuine platform dedicated to the investigation of bacterial proteins under reconstituted conditions, a very promising tool for the screening of new inhibitors against bacterial channels involved in drug resistance.

Integrated microdevice for long-term automated perfusion culture without shear stress and real-time electrochemical monitoring of cells.

Electrochemical techniques based on ultramicroelectrodes (UMEs) play a significant role in real-time monitoring of chemical messengers' release from single cells. Conversely, precise monitoring of cells in vitro strongly depends on the adequate construction of cellular physiological microenvironment. In this paper, we developed a multilayer microdevice which integrated high aspect ratio poly(dimethylsiloxane) (PDMS) microfluidic device for long-term automated perfusion culture of cells without shear stress and an independently addressable microelectrodes array (IAMEA) for electrochemical monitoring of the cultured cells in real time. Novel design using high aspect ratio between circular "moat" and ring-shaped micropillar array surrounding cell culture chamber combined with automated "circular-centre" and "bottom-up" perfusion model successfully provided continuous fresh medium and a stable and uniform microenvironment for cells. Two weeks automated culture of human umbilical endothelial cell line (ECV304) and neuronal differentiation of rat pheochromocytoma (PC12) cells have been realized using this device. Furthermore, the quantal release of dopamine from individual PC12 cells during their culture or propagation process was amperometrically monitored in real time. The multifunctional microdevice developed in this paper integrated cellular microenvironment construction and real-time monitoring of cells during their physiological process, and would possibly provide a versatile platform for cell-based biomedical analysis.

Solvatochromic dissociation of non-covalent fluorescent organic nanoparticles upon cell internalization.

Amorphous red-emitting materials involving solvatochromic small molecules have been processed by the reprecipitation method as non-doped nanospheres characterized by a remarkably low polydispersity. Their mean diameter could simply be tuned by the concentration of the organic solution giving rise to time-stable dispersion of 85-200 nm-sized nanoparticles. Time-resolved measurements performed on solid nanoparticles showed significant size-dependence effects of the emission lifetime and maxima evidencing populations with distinct molecular conformations. Nanoparticle internalization has proved successful in NIH-3T3 murine fibroblasts with normal toxicity effects after 48 h. Fluorescence confocal microscopy under one- and two-photon excitations revealed dual emission enabling localization of the organic material within the plasma membrane and the cytoplasm. Model experiments resorting to suspended artificial lipid bilayers allowed us to conclude on the dissolution of nanoparticles by the phospholipid membrane during the internalization process. They let us to assume that uptake of hydrophobic nanoparticles by living cells implies an endocytosis mechanism operating through the formation of plasmic vesicles.

Reusable Device for the Electrical Sensing of Red Blood Cells Rigidity Abnormalities, Based on A Reversible Microfluidic Assembly.

Combining microfluidic with sensors enables the development of smart analysis systems. Microelectrodes can be embedded within the microchannels network for electrical sensing, electrochemical analysis or impedance measurement. However, at the laboratory scale, the assembly between microfluidic network and electrical parts on the substrate remains an issue. This paper first discusses the principles of biosensing, and then proposes an original device integrating microfluidics with microelectrodes for the analysis of red blood cells transit in a structure mimicking micro-vascular flow. Some results concerning red blood cells discrimination of sickle cell disease are discussed with statistical analysis.Clinical relevance- This paper introduces a portable reusable device combining a microfluidic blood vessel mimicking network with microelectrodes for the biosensing of RBC.

Impact of pulsed electric fields and mechanical compressions on the permeability and structure of Chlamydomonas reinhardtii cells.

Current research findings clearly reveal the role of the microalga's cell wall as a key obstacle to an efficient and optimal compound extraction. Such extraction process is therefore closely related to the microalga species used. Effects of electrical or mechanical constraints on C. reinhardtii's structure and particularly its cell wall and membrane, is therefore investigated in this paper using a combination of microscopic tools. Membrane pores with a radius between 0.77 and 1.59 nm were determined for both reversible (5 kV∙cm-1) and irreversible (7 kV∙cm-1) electroporation with a 5 µs pulse duration. Irreversible electroporation with longer pulses (10 µs) lead to the entry of large molecules (at least 5.11 nm). Additionally, for the first time, the effect of pulsed electric fields on the cell wall was observed. The combined electrical and mechanical treatment showed a significant impact on the cell wall structure as observed under Transmission Electron Microscopy. This treatment permits the penetration of larger molecules (at least 5.11 nm) within the cell, shown by tracking the penetration of dextran molecules. For the first time, the size of pores on the cell membrane and the structural changes on the microalgae cell wall induced by electrical and mechanical treatments is reported.

Impedance spectroscopy study of the retinal pigment epithelium: Application to the monitoring of blue light exposure effect on A2E-loaded in-vitro cell cultures.

In age-related macular degeneration, the retinal pigment epithelium can be damaged by light acting on photosensitizers like N-retinylidene-N-retinylethanolamine (A2E). In this paper, the underlying cellular mechanism of lesion at the cell layer scale is analyzed by impedance spectroscopy. Retinal pigment epithelium (RPE) cells are cultured on top of custom-made electrodes capable of taking impedance measurements, with the help of a custom-made electronic setup but without the use of any chemical markers. An incubator is used to house the cells growing on the electrodes. An electrical model circuit is presented and linked to the constituents of the cell layer in which various electrical elements have been defined including a constant phase element (CPE) associated to the interface between the cell layer and the electrolyte. Their values are extracted from the fitted model of the measured impedance spectra. In this paper, we first investigate which parameters of the model can be analyzed independently. In that way, the parameter's evolution is examined with respect to two different targeted changes of the epithelium: 1. degradation of tight junctions between cells by extracellular calcium sequestration with Ethylenediaminetetraacetic acid (EDTA); 2. application of high amplitude short length electric field pulses. Based on the results obtained showing a clear relation between the model and the physiological state of the cell layer, the same procedure is applied to blue light exposure experiment. When A2E-loaded cells are exposed to blue light, the model parameters indicate, as expected, a clear degradation of the cell layer opposed to a relative stability of the not loaded ones.

A detailed in-vitro study of Retinal pigment epithelium's growth as seen from the perspective of impedance spectroscopy analysis.

In this paper, we present a method to assess growth and maturation phases of the Retinal Pigment Epithelium (RPE) in-vitro at the cell layer level using impedance spectroscopy measurements on platinum electrodes. We extracted relevant parameters from an electrical circuit model fitted with the measured spectra. Based on microscopic imaging, the growth state of an independent culture developing in the same conditions is used as reference. We show that the confluence point is identified from a graphical analysis of the spectra transition as well as by observing a reconstructed parameter representing the average capacitance of the cell layer. More generally, this work presents a detailed investigation on how cell culture's state relates with either model parameter analysis or with graphical analysis of the measured spectra over a wide frequency band. While applied to the RPE, this work is also suitable for the study of any kind of monolayer epithelial cells growth.

Characterization of red blood cell microcirculatory parameters using a bioimpedance microfluidic device.

This paper describes the use of a microfluidic device comprising channels with dimensions mimicking those of the smallest capillaries found in the human microcirculation. The device structure, associated with a pair of microelectrodes, provides a tool to electrically measure the transit time of red blood cells through fine capillaries and thus generate an electrical signature for red blood cells in the context of human erythroid genetic disorders, such as sickle cell disease or hereditary spherocytosis, in which red cell elasticity is altered. Red blood cells from healthy individuals, heated or not, and red blood cells from patients with sickle cell disease or hereditary spherocytosis where characterized at a single cell level using our device. Transit time and blockade amplitude recordings were correlated with microscopic observations, and analyzed. The link between the electrical signature and the mechanical properties of the red blood cells is discussed in the paper, with greater transit time and modified blockade amplitude for heated and pathological red blood cells as compared to those from healthy individuals. Our single cell-based methodology offers a new and complementary approach to characterize red cell mechanical properties in human disorders under flow conditions mimicking the microcirculation.

Characterization of sequentially-staged cancer cells using electrorotation.

The identification and separation of cells from heterogeneous populations is critical to the diagnosis of diseases. Label-free methodologies in particular have been developed to manipulate individual cells using properties such as density and morphology. The electrical properties of malignant cells, including the membrane capacitance and cytoplasmic conductivity, have been demonstrated to be altered compared to non-malignant cells of similar origin. Here, we exploit these changes to characterize individual cells in a sequentially-staged in vitro cancer model using electrorotation (EROT)-the rotation of a cell induced by a rotating electric field. Using a microfabricated device, a dielectrophoretic force to suspend cells while measuring their angular velocity resulting from an EROT force applied at frequencies between 3 kHz to 10 MHz. We experimentally determine the EROT response for cells at three stages of malignancy and analyze the resultant spectra by considering models that include the effect of the cell membrane alone (single-shell model) and the combined effect of the cell membrane and nucleus (double-shell model). We find that the cell membrane is largely responsible for a given cell's EROT response between 3 kHz and 10 MHz. Our results also indicate that membrane capacitance, membrane conductance, and cytoplasmic conductivity increase with an increasingly malignant phenotype. Our results demonstrate the potential of using electrorotation as a means making of non-invasive measurements to characterize the dielectric properties of cancer cells.

Reticulocyte and red blood cell deformation triggers specific phosphorylation events.

The capacity to undergo substantial deformation is a defining characteristic of the red blood cell (RBC), facilitating transit through the splenic interendothelial slits and microvasculature. Establishment of this remarkable property occurs during a process of reticulocyte maturation that begins with egress through micron-wide pores in the bone marrow and is completed within the circulation. The requirement to undertake repeated cycles of deformation necessitates that both reticulocytes and erythrocytes regulate membrane-cytoskeletal protein interactions in order to maintain cellular stability. In the absence of transcriptional activity, modulation of these interactions in RBCs is likely to be achieved primarily through specific protein posttranslational modifications, which at present remain undefined. In this study, we use high-throughput methods to define the processes that underlie the response to deformation and shear stress in both reticulocytes and erythrocytes. Through combination of a bead-based microsphiltration assay with phosphoproteomics we describe posttranslational modification of RBC proteins associated with deformation. Using microsphiltration and microfluidic biochip-based assays, we explore the effect of inhibiting kinases identified using this dataset. We demonstrate roles for GSK3 and Lyn in capillary transit and maintenance of membrane stability following deformation and show that combined inhibition of these kinases significantly decreases reticulocyte capacity to undergo repeated deformation. Finally, we derive a comprehensive and integrative phosphoproteomic dataset that provides a valuable resource for further mechanistic dissection of the molecular pathways that underlie the RBC's response to mechanical stimuli and for the study of reticulocyte maturation.

Understanding the mechanisms of lipid extraction from microalga Chlamydomonas reinhardtii after electrical field solicitations and mechanical stress within a microfluidic device.

One way envisioned to overcome part of the issues biodiesel production encounters today is to develop a simple, economically viable and eco-friendly process for the extraction of lipids from microalgae. This study investigates the lipid extraction efficiency from the microalga Chlamydomonas reinhardtii as well as the underlying mechanisms. We propose a new methodology combining a pulsed electric field (PEF) application and mechanical stresses as a pretreatment to improve lipid extraction with solvents. Cells enriched in lipids are therefore submitted to electric field pulses creating pores on the cell membrane and then subjected to a mechanical stress by applying cyclic pressures on the cell wall (using a microfluidic device). Results showed an increase in lipid extraction when cells were pretreated by the combination of both methods. Microscopic observations showed that both pretreatments affect the cell structure. Finally, the dependency of solvent lipid extraction efficiency with the cell wall structure is discussed.

Structural changes of Chlamydomonas reinhardtii cells during lipid enrichment and after solvent exposure.

Data are related to Confocal Laser Scanning Microscopy (CLSM) observations of lipid-enriched Chlamydomonas reinhardtii cells under different conditions. Firstly, the impact of stress conditions (nitrogen starvation) on the cell wall structure is assessed. Secondly is described the effect of solvents, in the context of lipid extraction, on the microalga's cell, particularly its lipid droplets, in the perspective of understanding the mechanisms behind solvent extraction of lipids. Furthermore, the role of the cell wall as a barrier to the solvent extraction action is highlighted.

Solid-State Nanopore Easy Chip Integration in a Cheap and Reusable Microfluidic Device for Ion Transport and Polymer Conformation Sensing.

Solid-state nanopores have a huge potential in upcoming societal challenging applications in biotechnologies, environment, health, and energy. Nowadays, these sensors are often used within bulky fluidic devices that can cause cross-contaminations and risky nanopore chips manipulations, leading to a short experimental lifetime. We describe the easy, fast, and cheap innovative 3D-printer-helped protocol to manufacture a microfluidic device permitting the reversible integration of a silicon based chip containing a single nanopore. We show the relevance of the shape of the obtained channels thanks to finite elements simulations. We use this device to thoroughly investigate the ionic transport through the solid-state nanopore as a function of applied voltage, salt nature, and concentration. Furthermore, its reliability is proved through the characterization of a polymer-based model of protein-urea interactions on the nanometric scale thanks to a hairy nanopore.