Label-free and noninvasive analysis of microorganism surface epistructures at the single-cell level.

Cell surface properties of microorganisms provide abundant information for their physiological status and fate choice. However, current methods for analyzing cell surface properties require labeling or fixation, which can alter the cell activity. This study establishes a label-free, rapid, noninvasive, and quantitative analysis of cell surface properties, including the presence and the dimension of epistructure, down to the single-cell level and at the nanometer scale. Simultaneously, electrorotation provides dielectric properties of intracellular contents. With the combined information, the growth phase of microalgae cells can be identified. The measurement is based on electrorotation of single cells, and an electrorotation model accounting for the surface properties is developed to properly interpret experimental data. The epistructure length measured by electrorotation is validated by scanning electron microscopy. The measurement accuracy is satisfactory in particular in the case of microscale epistructures in the exponential phase and nanoscale epistructures in the stationary phase. However, the measurement accuracy for nanoscale epistructures on cells in the exponential phase is offset by the effect of a thick double layer. Lastly, a diversity in epistructure length distinguishes exponential phase from stationary phase.

Dynamics of DNA Through Solid-state Nanopores Fabricated by Controlled Dielectric Breakdown.

Controlled dielectric breakdown (CDB) is gaining popularity for fabricating solid-state nanopores in situ with size control in a simple, low-cost, and scalable way. This technique could be used for a broad type of applications in the field of nucleic acid analysis and even for protein studies. In this work, we studied the entry and transport of double-stranded DNAs using a solid-state nanopore fabricated by CDB as a function of applied voltage for two different DNA lengths. We showed that the blockade rate increases exponentially with voltage up to 120 mV. The energy barrier depends on the chain length, and the dwell times decrease with applied voltage up to 120 mV. Moreover, no matter the chain length, it is possible to differentiate two families of blockade amplitudes, high and low ones, due to DNA folding.

Focus on using nanopore technology for societal health, environmental, and energy challenges.

With an increasing global population that is rapidly ageing, our society faces challenges that impact health, environment, and energy demand. With this ageing comes an accumulation of cellular changes that lead to the development of diseases and susceptibility to infections. This impacts not only the health system, but also the global economy. As the population increases, so does the demand for energy and the emission of pollutants, leading to a progressive degradation of our environment. This in turn impacts health through reduced access to arable land, clean water, and breathable air. New monitoring approaches to assist in environmental control and minimize the impact on health are urgently needed, leading to the development of new sensor technologies that are highly sensitive, rapid, and low-cost. Nanopore sensing is a new technology that helps to meet this purpose, with the potential to provide rapid point-of-care medical diagnosis, real-time on-site pollutant monitoring systems to manage environmental health, as well as integrated sensors to increase the efficiency and storage capacity of renewable energy sources. In this review we discuss how the powerful approach of nanopore based single-molecule, or particle, electrical promises to overcome existing and emerging societal challenges, providing new opportunities and tools for personalized medicine, localized environmental monitoring, and improved energy production and storage systems.

A versatile microfluidic tool for the 3D culture of HepaRG cells seeded at various stages of differentiation.

The development of livers-on-a-chip aims to provide pharmaceutical companies with reliable systems to perform drug screening and toxicological studies. To that end, microfluidic systems are engineered to mimic the functions and architecture of this organ. In this context we have designed a device that reproduces series of liver microarchitectures, each permitting the 3D culture of hepatocytes by confining them to a chamber that is separated from the medium conveying channel by very thin slits. We modified the structure to ensure its compatibility with the culture of hepatocytes from different sources. Our device was adapted to the migratory and adhesion properties of the human HepaRG cell line at various stages of differentiation. Using this device, it was possible to keep the cells alive for more than 14 days, during which they achieved a 3D organisation and acquired or maintained their differentiation into hepatocytes. Albumin secretion as well as functional bile canaliculi were confirmed on the liver-on-a-chip. Finally, an acetaminophen toxicological assay was performed. With its multiple micro-chambers for hepatocyte culture, this microfluidic device architecture offers a promising opportunity to provide new tools for drug screening applications.

Selective target protein detection using a decorated nanopore into a microfluidic device.

Solid-state nanopores provide a powerful tool to electrically analyze nanoparticles and biomolecules at single-molecule resolution. These biosensors need to have a controlled surface to provide information about the analyte. Specific detection remains limited due to nonspecific interactions between the molecules and the nanopore. Here, a polymer surface modification to passivate the membrane is performed. This functionalization improves nanopore stability and ionic conduction. Moreover, one can control the nanopore diameter and the specific interactions between protein and pore surface. The effect of ionic strength and pH are probed. Which enables control of the electroosmotic driving force and dynamics. Furthermore, a study of polymer chain structure and permeability in the pore are carried out. The nanopore chip is integrated into a microfluidic device to ease its handling. Finally, a discussion of an ionic conductance model through a permeable crown along the nanopore surface is elucidated. The proof of concept is demonstrated by the capture of free streptavidin by the biotins grafted into the nanopore. In the future, this approach could be used for virus diagnostic, nanoparticle or biomarker sensing.

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.

Electrorotation of single microalgae cells during lipid accumulation for assessing cellular dielectric properties and total lipid contents.

Photosynthetic microalgae not only perform fixation of carbon dioxide but also produce valuable byproducts such as lipids and pigments. However, due to the lack of effective tools for rapid and noninvasive analysis of microalgal cellular contents, the efficiency of strain screening and culture optimizing is usually quite low. This study applied single-cell electrorotation on Scenedesmus abundans to assess cellular dielectric properties during lipid accumulation and to promptly quantify total cellular contents. The experimental electrorotation spectra were fitted with the double-shell ellipsoidal model, which considered varying cell wall thickness, to obtain the dielectric properties of cellular compartments. When the amount of total lipids increased from 15.3 wt% to 33.8 wt%, the conductivity and relative permittivity of the inner core (composed of the cytoplasm, lipid droplets, and nucleus) decreased by 21.7% and 22.5%, respectively. These dielectric properties were further used to estimate the total cellular lipid contents by the general mixing formula, and the estimated values agreed with those obtained by weighing dry biomass and extracted lipids with an error as low as 0.22 wt%. Additionally, the conductivity and relative permittivity of cell wall increased during nitrogen-starvation conditions, indicating the thickening of cell wall, which was validated by the transmission electron microscopy.

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.

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.

Brownian Motion and Large Electric Polarizabilities Facilitate Dielectrophoretic Capture of Sub-200 nm Gold Nanoparticles in Water.

Dielectrophoresis can move small particles using the force resulting from their polarization in a divergent electric field. In liquids, it has most often been applied to micrometric objects such as biological cells or latex microspheres. For smaller particles, the dielectrophoretic force becomes very small and the phenomenon is furthermore perturbed by Brownian motion. Whereas dielectrophoresis has been used for assembly of superstructures of nanoparticles and for the detection of proteins and nucleic acids, the mechanisms underlying DEP of such small objects require further study. This work presents measurements of the alternating-current (AC) dielectrophoretic response of gold nanoparticles of less than 200 nm diameter in water. An original dark-field digital video-microscopic method was developed and used in combination with a microfluidic device containing transparent thin-film electrodes. It was found that the dielectrophoretic force is only effective in a small zone very close to the tip of the electrodes, and that Brownian motion actually facilitates transport of particles towards this zone. Moreover, the fact that particles as small as 80 nm are still efficiently captured in our device is not only due to Brownian transport but also to an effective polarizability that is larger than what would be expected on basis of current theory for a sphere in a dielectric medium.

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.

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.

A microfluidic approach to study the effect of mechanical stress on erythrocytes in sickle cell disease.

The human red blood cell is a biconcave disc of 6-8 × 2 µm that is highly elastic. This capacity to deform enables it to stretch while circulating through narrow capillaries to ensure its main function of gas exchange. Red cell shape and deformability are altered in membrane disorders because of defects in skeletal or membrane proteins affecting protein-protein interactions. Red cell properties are also altered in other pathologies such as sickle cell disease. Sickle cell disease is a genetic hereditary disorder caused by a single point mutation in the ß-globin gene generating sickle haemoglobin (HbS). Hypoxia drives HbS polymerisation that is responsible for red cell sickling and reduced deformability. The main clinical features of sickle cell disease are vaso-occlusive crises and haemolytic anaemia. Foetal haemoglobin (HbF) inhibits HbS polymerisation and positively impacts red cell survival in the circulation but the mechanism through which it exerts this action is not fully characterized. In this study, we designed a microfluidic biochip mimicking the dimensions of human capillaries to measure the impact of repeated mechanical stress on the survival of red cells at the single cell scale under controlled pressure. We show that mechanical stress is a critical parameter underlying intravascular haemolysis in sickle cell disease and that high intracellular levels of HbF protect against lysis. The biochip is a promising tool to address red cell deformability in pathological situations and to screen for molecules positively impacting this parameter in order to improve red cell survival in the circulation.

From current trace to the understanding of confined media.

Nanopores constitute devices for the sensing of nano-objects such as ions, polymer chains, proteins or nanoparticles. We describe what information we can extract from the current trace. We consider the entrance of polydisperse chains into the nanopore, which leads to a conductance drop. We describe the detection of these current blockades according to their shape. Finally, we explain how data analysis can be used to enhance our understanding of physical processes in confined media.

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.

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.

Functionalized Solid-State Nanopore Integrated in a Reusable Microfluidic Device for a Better Stability and Nanoparticle Detection.

Electrical detection based on single nanopores is an efficient tool to detect biomolecules, particles and study their morphology. Nevertheless the surface of the solid-state membrane supporting the nanopore should be better controlled. Moreover, nanopore should be integrated within microfluidic architecture to facilitate control fluid exchanges. We built a reusable microfluidic system integrating a decorated membran, rendering the drain and refill of analytes and buffers easier. This process enhances strongly ionic conductance of the nanopore and its lifetime. We highlight the reliability of this device by detecting gold nanorods and spherical proteins.

Electric pulses: a flexible tool to manipulate cytosolic calcium concentrations and generate spontaneous-like calcium oscillations in mesenchymal stem cells.

Human adipose mesenchymal stem cells (haMSCs) are multipotent adult stem cells of great interest in regenerative medicine or oncology. They present spontaneous calcium oscillations related to cell cycle progression or differentiation but the correlation between these events is still unclear. Indeed, it is difficult to mimic haMSCs spontaneous calcium oscillations with chemical means. Pulsed electric fields (PEFs) can permeabilise plasma and/or organelles membranes depending on the applied pulses and therefore generate cytosolic calcium peaks by recruiting calcium from the external medium or from internal stores. We show that it is possible to mimic haMSCs spontaneous calcium oscillations (same amplitude, duration and shape) using 100 µs PEFs or 10 ns PEFs. We propose a model that explains the experimental situations reported. PEFs can therefore be a flexible tool to manipulate cytosolic calcium concentrations. This tool, that can be switched on and off instantaneously, contrary to chemicals agents, can be very useful to investigate the role of calcium oscillations in cell physiology and/or to manipulate cell fate.

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.