Application of droplet-based microfluidics for the screening of microbial libraries is one of the important ongoing developments in functional genomics/metagenomics. In this article, we propose a new method that can be employed for high-throughput profiling of cell growth. It consists of light-driven labelling droplets that contain growing cells directly in a microfluidics observation chamber, followed by recovery of the labelled cells. This method is based on intracellular expression of green-to-red switchable fluorescent proteins. The proof of concept is established here for two commonly used biological models, E. coli and S. cerevisiae. Growth of cells in droplets was monitored under a microscope and, depending on the targeted phenotype, the fluorescence of selected droplets was switched from a "green" to a "red" state. Red fluorescent cells from labelled droplets were then successfully detected, sorted with the Fluorescence Activated Cell Sorting machine and recovered. Finally, the application of this method for different kind of screenings, in particular of metagenomic libraries, is discussed and this idea is validated by the analysis of a model mini-library.

Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA analysis in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification. This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions with high throughput processing up to 120 μL of DNA dilution at flow rates ranging from 1 to 5 μL/min without compromising performance. The fluidized bed was 20–50% more efficient than a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based microarray and tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostics.

major challenge in cancer research is the complexity of the tumor microenvironment, which includes the host immunological setting. Inspired by the emerging technology of organ-on-chip, we achieved 3D co-cultures in microfluidic devices (integrating four cell populations: cancer, immune, endothelial, and fibroblasts) to reconstitute ex vivo a human tumor ecosystem (HER2+ breast cancer). We visualized and quantified the complex dynamics of this tumor-on-chip, in the absence or in the presence of the drug trastuzumab (Herceptin), a targeted antibody therapy directed against the HER2 receptor. We uncovered the capacity of the drug trastuzumab to specifically promote long cancer-immune interactions (>50 min), recapitulating an anti-tumoral ADCC (antibody-dependent cell-mediated cytotoxicity) immune response. Cancer-associated fibroblasts (CAFs) antagonized the effects of trastuzumab. These observations constitute a proof of concept that tumors-on-chip are powerful platforms to study ex vivo immunocompetent tumor microenvironments, to characterize ecosystem-level drug responses, and to dissect the roles of stromal components.

Droplet microfluidics allows the implementation of several biochemical and bioanalytical protocols in microfluidic devices. However, this integration typically requires the use of microfluidic valves, which allow the control of different operations on the same channels network. Unfortunately, the valves commercially available are almost exclusively suitable for-single phase flow, and only one valve system compatible with multi-phase systems has been presented so far in the literature. We present here a new pinch-like valve that withstands high pressures (tested up to 4 bar), allows droplet manipulation and combines the advantages of conventional pinch valves regarding the absence of dead volume, while making negligible the backflow generated (less than 2 nL). The conception and design of the presented valve, based on numerical simulations, is performed by an optimized microfabrication strategy, combining PDMS soft-lithography and 3D printing technologies. The valves had been tested regarding their mechanical stability and proving their capability of allowing droplets passing through them, without any perturbation. Finally, the very small back-flow volume generated during the actuation, has been exploited for various applications in droplets microfluidics, like droplet handling, droplet-on-demand generation and sorting.

In this study, single-step magnetic-beads based immunoassays was developed and for the first time adapted to microfluidic droplet operation for sequential determination of well-established amyloid-β peptide biomarkers (i.e. monomeric Aβ 1–42 and Aβ 1–40) for molecular diagnosis of Alzheimer’s disease (AD). With the developed sandwich assay protocol, the capture antibodies (i.e. monoclonal anti-Aβ antibodies that are specific for N-terminus of Aβ 1–42 and Aβ 1–40) grafted onto magnetic beads and the detection antibodies (i.e. beta amyloid 1–16 monoclonal antibody labeled with horseradish peroxidase) can simultaneously bind to monomeric β peptides in a single step. Aβ 1–42 and Aβ 1–40 in cerebrospinal fluid (CSF) samples were successfully detected using the developed batchwise immunoassay approach. With the aim at expanding the spectrum of traced Aβ peptides beyond Aβ 1–42 and Aβ 1–40 for more precise diagnostics of AD, the developed sandwich assay was for the first time coupled as a downstream module to peptide fractionation and collection using capillary isoelectric focusing. Demonstration of this methodological combination was carried out with Aβ 1–40, Aβ 1–40 and Aβ 5–40. The immunoassay in batchwise format was downscaled into a purpose-made microfluidic droplet platform allowing significant sample volume reduction and higher throughput. Using a series of 4 programmable magnetic tweezers to manipulate a train of nano-scale confined droplets containing magnetic beads, sample, washing and detection solutions, a sequence of 8 analyses could be realized within 45 min. This droplet based immunoassay was realized in a dedicated platform integrating an in-house-made light emitting diode (LED)-based fluorescent detector, replacing conventional microscopic setup so as to significantly reduce the construction cost and simplify the detection protocol. Using this microfluidic configuration coupled with LED-based detection, information on both Aβ 1–42 and Aβ 1–40 concentrations can be collected in a single sequence with less than 1 μL of sample.

The vast majority of current microfluidic devices are produced using soft lithography, a technique with strong limitations regarding the fabrication of three-dimensional architectures. Additive manufacturing holds great promises to overcome these limitations, but conventional machines still lack the resolution required by most microfluidic applications. 3D printing machines based on two-photon lasers, in contrast, have the needed resolution but are too limited in speed and size of the global device. Here we demonstrate how the resolution of conventional stereolithographic machines can be improved by a direct programming of the laser path and can contribute to bridge the gap between the two above technologies, allowing the direct printing of features between 10 and 100 μm, corresponding to a large fraction of microfluidic applications. This strategy allows to achieve resolutions limited only by the physical size of the laser beam, decreasing by a factor at least 2× the size of the smallest features printable, and increasing their reproducibility by a factor 5. The approach was applied to produce an open microfluidic device with the reversible seal, integrating periodical patterns using the simple motifs, and validated by the fabrication of a deterministic lateral displacement particles sorting device. The sorting of polystyrene beads (diameter: 20 μm and 45 μm) was achieved with a specificity >95%, comparable with that achieved with arrays prepared by microlithography.

A microfluidic microreactor for trypsin mediated transthyretin (TTR) digestion has been developed as a step towards the elaboration of a fully integrated microdevice for the detection of a rare and disabling disease, the familial transthyretin amyloidosis (ATTR) which is related to specific TTR mutations. Therefore, an enzymatic microreactor coupled to an analytical step able to monitor the mutation of TTR on specific peptide fragments would allow an accurate monitoring of the treatment efficiency of ATTR. In this study, two types of immobilized trypsin microreactors have been investigated: a new miniaturized, microfluidic fluidized bed packed with trypsin functionalized magnetic particles (MPs), and a thiol-ene (TE) monolith-based chip. Their performances were first demonstrated with N-benzoyl-dl-arginine-4-nitroanilide hydrochloride BApNA, a low molecular weight substrate. High reaction yields (75.2%) have been reached within 0.6 min for the TE-based trypsin microreactor, while a lower yield (12.4%) was obtained for the micro-fluidized bed within a similar residence time. Transposition of the optimized conditions, developed with BApNA, to TTR digestion in the TE-based trypsin microreactor was successfully performed. We demonstrated that the TE-chip can achieve an efficient and reproducible digestion of TTR. This has been assessed by MS detection. In addition, TTR hydrolysis led to the production of a fragment of interest allowing the therapeutic follow-up of more than twenty possible ATTR mutations. High sequence coverage (90%), similar to those obtained with free trypsin, was achieved in a short time (2.4 min). Repeated experiments showed good reproducibility (RSD = 6.8%). These promising results open up the route for an innovative treatment follow-up dedicated to ATTR.

Fluorescence measurement is the main technology for post-amplification DNA detection in automated systems. Direct electrical reading of DNA concentration in solution could be an interesting alternative to go toward more miniaturized or less expensive devices, in particular in the pathogen detection field. Here we present the detection of short bacterial biomarkers with a direct impedancemetric measurement, within solutions of amplified and elongated DNA sequences in a microchannel. This technology relies on the electrohydrodynamic instability occurring in solutions of long charged macromolecules in a strong electric field. This instability specifically induces the aggregation of long DNAs and triggers conductivity variations that can be monitored by on-contact conductometry. An innovative isothermal amplification and elongation strategy was developed, combining SDA and HRCA reactions, in order to yield long DNAs suitable to be detected by the above principle, from a dilute initial DNA target. In contrast with previous label-free detection methods, this new strategy is very robust to matrix effects, thanks to the unique molecular weight dependence of the instability, coupled with this specific DNA amplification strategy. We demonstrate the detection of a 1 pM gene sequence specific to Staphylococcus aureus, in a portable system.

Tumor initiation and growth is associated with significant changes in the surrounding tissue. During carcinoma progression, a global stiffening of the extracellular matrix is observed and is interpreted as a signature of aggressive invasive tumors. However, it is still unknown whether this increase in matrix rigidity promotes invasion and whether this effect is constant along the course of invasion. Here we have developed a biomimetic in vitro assay that enabled us to address the question of the importance of tissue rigidity in the chronology of tumor invasion. Using low concentrations of the sugar threose, we can effectively stiffen reconstituted collagen I matrices and control the stiffening in time with no direct effect on residing cells. Our findings demonstrate that, depending on the timing of its stiffening, the extracellular matrix could either inhibit or promote cancer cell invasion and subsequent metastasis: while matrix stiffening after the onset of invasion promotes cancer cell migration and tumor spreading, stiff matrices encapsulate the tumor at an early stage and prevent cancer cell invasion. Our study suggests that adding a temporal dimension in in vitro models to analyze biological processes in four dimensions is necessary to fully capture their complexity.

The success of microfluidic immunocapture based on magnetic beads depends primarily on a sophisticated microscale separation system and on the quality of the magnetic immunosorbent. A microfluidic chip containing a magnetically stabilized fluidized bed (μMSFB), developed for the capture and on-chip amplification of bacteria, was recently described by Pereiro et al.. The present work shows the thorough development of anti-Salmonella magnetic immunosorbents with the optimal capture efficiency and selectivity. Based on the corresponding ISO standards, these parameters have to be high enough to capture even a few cells of bacteria in a proper aliquot of sample, e.g. milk. The selection of specific anti-Salmonella IgG molecules and the conditions for covalent bonding were the key steps in preparing an immunosorbent of the desired quality. The protocol for immunocapturing was first thoroughly optimized and studied in a batchwise arrangement, and then the carrier was integrated into the μMSFB chip. The combination of the unique design of the chip (guaranteeing the collision of cells with magnetic beads) with the advanced immunosorbent led to a Salmonella cell capture efficiency of up to 99%. These high values were achieved repeatedly even in samples of milk differing in fat content. The rate of nonspecific capture of Escherichia coli (i.e. the negative control) was only 2%.

An experimental design is described for optimization of the microscopic morphology of a methacrylate monolith that was elaborated for chromatographic separation of radionuclides in nitric acid media. This paper presents a systematic study of the synthesis of the polymeric porous monolith poly(ethylene glycol dimethacrylate-co-allyl methacrylate) used as solid-phase support and a post-functionalization of the monolith in microsystem with tributyl phosphate extractant. Polymerization time and chemical composition of the polymerizable mixture that comprises water, 1,4-butanediol, 1-propanol, monomers were chosen as the most relevant experimental factors of the photochemical process. Using the globules area as a significant response of an experimental design, the monolith morphology can be predicted. A new versatile and robust impregnation process was developed in microsystem. The designed micro chromatographic system showed a good resistance in concentrated nitric acid and a great loading capacity compared to commercially available solution (150 mg U versus 75 mgU/g resin).

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

High electromechanical coupling is critical to perform effective conversion between mechanical and electrical energy for various applications of electrostrictive polymers. Herein, a giant electrostriction effect is reported in liquid crystalline graphene doped dielectric elastomers. The materials are formulated by an original phase transfer method which allows the solubilization of graphenic monolayers in non-polar solvents. Dielectric spectroscopy is combined with tensile test devices to measure the true electrostriction coefficients with differentiating the Maxwell stress effect. Because of their unique liquid crystal structure, the resultant composites show a giant relative permittivity and ultralarge electrostriction coefficient. This work offers a promising pathway to design novel high performance electrostrictive polymer composites as well as to provide insights into mechanisms of true electrostriction in electrically …

We present electrostrictive materials with excellent properties for vibrational energy harvesting applications. The developed materials consist of a porous carbon black composite, which is processed using water-in-oil emulsions. In combination with an insulating layer, the investigated structures exhibit a high effective relative dielectric permittivity (up to 182 at 100 Hz) with very low effective conductivity (down to 2.53 10− 8 S m− 1). They can generate electrical energy in response to mechanical vibrations with a power density of 0.38 W m− 3 under an applied bias electric field of 32 V. They display figures or merit for energy harvesting applications well above reference polymer materials in the field, including fluorinated co-and ter-polymers synthetized by heavy chemical processes. The production process of the present materials is based on non hazardous and low-cost chemicals. The soft dielectric materials are …

Drying graphene oxide (GO) films are subject to extensive wrinkling, which largely affects their final properties. Wrinkles were shown to be suitable in biotechnological applications; however, they negatively affect the electronic properties of the films. Here, we report on wrinkle tuning and patterning of GO films under stress-controlled conditions during drying. GO flakes assemble at an air–solvent interface; the assembly forms a skin at the surface and may bend due to volume shrinkage while drying. We applied a modification of evaporative lithography to spatially define the evaporative stress field. Wrinkle alignment is achieved over cm2 areas. The wavelength (i.e., wrinkle spacing) is controlled in the μm range by the film thickness and GO concentration. Furthermore, we propose the use of nanoparticles to control capillary forces to suppress wrinkling. An example of a controlled pattern is given to elucidate the …

Recent advances in the field of microelectromechanical systems (MEMS) have generated great interest in the substitution of inorganic microcantilevers by organic ones, due to their low cost, high flexibility and a simplified fabrication by means of printing methods. Here, we present the integration of electrostrictive nanocomposites into organic microcantilever resonators specifically designed for mechanical energy harvesting from ambient vibrations. Strain sensitive nanocomposite materials composed of reduced graphene oxide (rGO) dispersed in polydimethylsiloxane (PDMS) are integrated into all-organic MEMS by means of an innovative low-cost and environment friendly process by combining printing techniques and xurography. Static tests of the electrostrictive nanocomposite with 3.7 wt% rGO show good performances with variations of capacitance that exceeds 4% for strain values lower than 0.55% as the …

High electromechanical coupling is critical to perform effective conversion between mechanical and electrical energy for various applications of electrostrictive polymers. Herein, a giant electrostriction effect is reported in liquid crystalline graphene-doped dielectric elastomers. The materials are formulated by a phase-transfer method which allows the solubilization of graphenic monolayers in nonpolar solvents. Dielectric spectroscopy is combined with tensile test devices to measure the true electrostriction coefficients with differentiating the Maxwell stress effect. Because of their liquid crystal structure, the resultant composites show an ultralarge electrostriction coefficient (∼10–14 m2/V2 at 0.1 Hz) coupled with good reproducibility during cycles at high deformation rates. This work offers a promising pathway to design high-performance electrostrictive polymer composites as well as to provide insights into mechanisms …

We study the flow of suspensions of non-Brownian particles dispersed into a Newtonian solvent. Combining capillary rheometry and conventional rheometry, we evidence a succession of two shear thinning regimes separated by a shear thickening one. Through X-ray radiography measurements, we show that during each of those regimes, the flow remains homogeneous and does not involve particle migration. Using a quartz-tuning fork based atomic force microscope, we measure the repulsive force profile and the microscopic friction coefficient μ between two particles immersed into the solvent, as a function of normal load. Coupling measurements from those three techniques, we propose that (1) the first shear-thinning regime at low shear rates occurs for a lubricated rheology and can be interpreted as a decrease of the effective volume fraction under increasing particle pressures, due to short-ranged repulsive …