Search

For over 30 years, carbon fiber microelectrodes have been the gold standard for measurements related to exocytosis and more generally to the processes taking place at the synaptic level. However, this method has a low throughput and molecules can escape detection due to the featureless nature of the planar microelectrodes it uses. Here we present a new electrochemical sensor that addresses these limitations. It is based on insulated protruding volcano-shaped tips of 2 mu m in diameter housing two individually addressable microelectrodes. The sensor enables volume confined and parallelizable recordings of exocytosis from adherent cells. Exocytotic releases from PC12 cells measured by amperometry on our device have quantal size in agreement with commonly admitted values but happen on a much smaller time scale; mostly within half a millisecond. We demonstrate that this faster kinetics must involve a faster vesicle fusion mechanism and is plausibly due to perturbation of the plasma membrane brought by the topography of our sensor. This suggests that exocytosis kinetics may be manipulated by the adequate substrate geometry, which opens up promising new leads of investigation in the study of synaptic processes.

The generation of single-cell biological assays has become a strategic need for fundamental and clinical studies. Droplet microfluidics being sensitive, high throughput, and low cost, has been widely adopted for single-cell assays. However, precise manipulation of single cells using droplet microfluidics is still limited by the intrinsic randomness during single cell encapsulation, known as the Poisson limitation. It results in ambiguous single-cell assays with considerable cell losses. Here we present a novel cell-triggered splitting (CTS) method for deterministic and passive single cell encapsulation into droplets. We demonstrated the high efficacy (negligible cell loss) and high specificity (high cell loading percentage) of this method. With a simple working principle, this passive and label-free method reaches an unprecedented throughput of more than 3k Hz for droplet sorting, corresponding to 22,000 cell-loaded droplets per minute with less than 3% false events (empty droplets or doublets), a 100-fold improvement compared to existing label-free active droplet sorting methods. This asynchronous method requires no flow tuning and provides a general solution to different application scenarios, from high throughput encapsulation to manipulation of small cell populations. This method also applies to the encapsulation of other deformable particles such as hydrogel beads. The novel versatile tool may impact applications in single-cell sequencing, cell-cell/cell-bead interaction, and rare cell isolation. This passive and deterministic single-cell encapsulation method provides a practical solution to overcome the long-existing difficulty associated with the Poisson limit.

The understanding of cell-cell and cell-matrix interactions via receptor and ligand binding relies on our ability to study the very first events of their contact. Of particular interest is the interaction between a T cell receptor and its cognate peptide-major histocompatibility complex. Indeed analyzing their binding kinetics and cellular avidity in large-scale low-cost and fast cell sorting would largely facilitate the access to cell-based cancer immunotherapies. We thus propose a microfluidic tool able to independently control two types of micro-sized objects, put them in contact for a defined time and probe their adhesion state. The device consists in hydrodynamic traps holding the first type of cells from below against the fluid flow, and a dielectrophoretic system to force the second type of object to remain in contact to the first one. First the device is validated by performing an adhesion frequency assay between fibroblasts and fibronectin coated bead. Then, a study is conducted on the modification of the cellular environment to match the dielectrophoretic technology requirements without modifying the cells viability and interaction functionalities. Finally, we demonstrate the capability of the developed device to put cancer cells and a population of T cells in contact and show the discrimination between specific and non specific interactions based on pairs lifetime.

We present a novel concept for the controlled trapping and releasing of beads and cells in a PDMS microfluidic channel without obstacles present around the particle or in the channel. The trapping principle relies on a two-level microfluidic configuration: a top main PDMS channel interconnected to a buried glass microchannel using round vias. As the fluidic resistances rule the way the liquid flows inside the channels, particles located in the streamlines passing inside the buried level are immobilized by the round via with a smaller diameter, leaving the object motionless in the upper PDMS channel. The particle is maintained by the difference of pressure established across its interface and acts as an infinite fluidic resistance, virtually cancelling the subsequent buried fluidic path. The pressure is controlled at the outlet of the buried path and three modes of operation of a trap are defined: idle, trapping and releasing. The pressure conditions for each mode are defined based on the hydraulic–electrical circuit equivalence. The trapping of polystyrene beads in a compact array of 522 parallel traps controlled by a single pressure was demonstrated with a trapping efficiency of 94%. Pressure conditions necessary to safely trap cells in holes of different diameters were determined and demonstrated in an array of 25 traps, establishing the design and operation rules for the use of planar hydrodynamic traps for biological assays., A new process for the fabrication of two superposed layers of microfluidic channels connected by vias is used to trap and release particles in a transparent chip. Parallel manipulation of beads is studied and the rules for cell trapping are defined.

Continuous fluidic sampling systems allow collection of brain biomarkers in vivo. Here, we propose a new sampling paradigm, Droplet on Demand (DoD), implemented in a microfabricated neural probe. It allows sampling droplets loaded with molecules from the brain extracellular fluid punctually, without the long transient equilibration periods typical of continuous methods. It uses an accurate fluidic sequence and correct operation is verified by the embedded electrodes. As a proof of concept, we demonstrated the application of this novel approach in vitro and in vivo, to collect glucose in the brain of mice, with a temporal resolution of 1-2 minutes and without transient regime. Absolute quantification of the glucose level in the samples was performed by direct infusion nanoelectrospray ionization Fourier transform mass spectrometry (nanoESI-FTMS). By adjusting the diffusion time and the perfusion volume of DoD, the fraction of molecules recovered in the samples can be tuned to mirror the tissue concentration at accurate points in time. This makes quantification of biomarkers in the brain possible within acute experiments of only 20 to 120 minutes. DoD provides a complementary tool to continuous microdialysis and push-pull sampling probes. The advances allowed by DoD will benefit quantitative molecular studies in the brain, namely for molecules involved in volume transmission or for protein aggregates that form in neurodegenerative diseases over long periods.Abstract Figure

Droplet breakup is an important phenomenon in the field of microfluidics to generate daughter droplets. In this work, a novel breakup regime in the widely studied T-junction geometry is reported, where the pinch-off occurs laterally in the two outlet channels, leading to the formation of three daughter droplets, rather than at the center of the junction for conventional T-junctions which leads to two daughter droplets. It is demonstrated that this new mechanism is driven by surface tension, and a design rule for the T-junction geometry is proposed. A model for low values of the capillary number $Ca$ is developed to predict the formation and growth of an underlying carrier fluid pocket that accounts for this lateral breakup mechanism. At higher values of $Ca$, the conventional regime of central breakup becomes dominant again. The competition between the new and the conventional regime is explored. Altogether, this novel droplet formation method at T-junction provides the functionality of alternating droplet size and composition, which can be important for the design of new microfluidic tools.

Volcano-shaped microelectrodes (nanovolcanoes) functionalized with nanopatterned self-assembled monolayers have recently been demonstrated to report cardiomyocyte action potentials after gaining spontaneous intracellular access. These nanovolcanoes exhibit recording characteristics similar to those of state-of-the-art micro-nanoelectrode arrays that use electroporation as an insertion mechanism. In this study, we investigated whether the use of electroporation improves the performance of nanovolcano arrays in terms of action potential amplitudes, recording durations, and yield. Experiments with neonatal rat cardiomyocyte monolayers grown on nanovolcano arrays demonstrated that electroporation pulses with characteristics derived from analytical models increased the efficiency of nanovolcano recordings, as they enabled multiple on-demand registration of intracellular action potentials with amplitudes as high as 62 mV and parallel recordings in up to ~76% of the available channels. The performance of nanovolcanoes showed no dependence on the presence of functionalized nanopatterns, indicating that the tip geometry itself is instrumental for establishing a tight seal at the cell–electrode interface, which ultimately determines the quality of recordings. Importantly, the use of electroporation permitted the recording of attenuated cardiomyocyte action potentials during consecutive days at identical sites, indicating that nanovolcano recordings are nondestructive and permit long-term on-demand recordings from excitable cardiac tissues. Apart from demonstrating that less complex manufacturing processes can be used for next-generation nanovolcano arrays, the finding that the devices are suitable for performing on-demand recordings of electrical activity from multiple sites of excitable cardiac tissues over extended periods of time opens the possibility of using the devices not only in basic research but also in the context of comprehensive drug testing. Arrays of microelectrodes allow researchers to record electrophysiological data from healthy, living cells for extended durations. Such measurements, which can offer valuable insights into phenomena like neuronal firing or muscle excitation, are typically cumbersome to collect. Researchers led by Benoit Desbiolles of the Ecole Polytechnique Federale de Lausanne in Switzerland have devised a promising alternative, based on so-called ‘nanovolcanoes’. These hollow, conical electrodes form a seal with cell membranes. By then applying a controlled electric field across the membrane, the researchers opened up transient pores in the membrane, permitting the recording of membrane voltage changes. This enabled them to reliably monitor the electrophysiological activity of more than a dozen cells in parallel with a single device and to perform transmembrane voltage recordings at will over the course of a two-day period without injuring the cells.

Atomic force microscopy based approaches have led to remarkable advances in the field of mechanobiology. However, linking the mechanical cues to biological responses requires complementary techniques capable of recording these physiological characteristics. In this study, we present an instrument for combined optical, force, and electrical measurements based on a novel type of scanning probe microscopy cantilever composed of a protruding volcano-shaped nanopatterned microelectrode (nanovolcano probe) at the tip of a suspended microcantilever. This probe enables simultaneous force and electrical recordings from single cells. Successful impedance measurements on mechanically stimulated neonatal rat cardiomyocytes in situ were achieved using these nanovolcano probes. Furthermore, proof of concept experiments demonstrated that extracellular field potentials (electrogram) together with contraction displacement curves could simultaneously be recorded. These features render the nanovolcano probe especially suited for mechanobiological studies aiming at linking mechanical stimuli to electrophysiological responses of single cells.

Differentiation of stem cells into mature cells through the use of physical approaches is of great interest. Here, we prepared smart nanoenvironments by cell-imprinted substrates based on chondrocytes, tenocytes, and semifibroblasts as templates and demonstrated their potential for differentiation, redifferentiation, and transdifferentiation. Analysis of shape and upregulation/downregulation of specific genes of stem cells, which were seeded on these cell-imprinted substrates, confirmed that imprinted substrates have the capability to induce specific shapes and molecular characteristics of the cell types that were used as templates for cell-imprinting. Interestingly, immunofluorescent staining of a specific protein in chondrocytes (i.e., collagen type II) confirmed that adipose-derived stem cells, semifibroblasts, and tenocytes can acquire the chondrocyte phenotype after a 14 day culture on chondrocyte-imprinted substrates. In summary, we propose that common polystyrene tissue culture plates can be replaced by this imprinting technique as an effective and promising way to regulate any cell phenotype in vitro with significant potential applications in regenerative medicine and cell-based therapies.

Here we demonstrate enhanced proton conduction through polyelectrolyte matrices comprising nanopeapods of phosphotungstic acid (PWA) filled carbon nanotubes (CNTs). The ionic nanopeapods were found to provide rapid proton conduction pathways to design nanocomposite proton exchange membranes (PEMs) for high-performance fuel cell applications. Nanopeapod (0.5 wt%) incorporated Nafion-based PEMs offer improved proton conductivity, especially at elevated temperatures and low-humidity (0.202 S cm−1 compared with 0.132 S cm−1 for recast Nafion membrane at 90 °C), and about four times higher maximum power density at 40% R.H. and 120 °C (302 mW cm−2vs. 84 mW cm−2 for recast Nafion).

Despite its simplicity, which makes it the most commonly used bioprinting method today, extrusion-based bioprinting suffers from its inability to reproduce the complex tissue architecture found in organs. Generally, this printing method allows for the dispensing of solutions of a predefined cell concentration through a rudimentary needle. Moreover, to avoid cell lysis in the dispensing needle, which is detrimental to the viability of the printed tissue, as well as cell loss in dead volumes of tubing, thereby increasing the cost of printing tissue, a common strategy has been to print with cell concentrations much lower in comparison to the concentrations found in living tissues. As a result, cell-to-cell distance is increased in the dispensed samples impairing communication through cytokines. Here, we present a microfluidic-based print head capable of modulating the printed cell concentration in real-time. This device allows bioprinting at high cell concentrations by concentrating and dispensing fibroblasts at concentrations up to 10 million cells∙mL−1. We also demonstrate that this device can be used to print bladder organoids. As the cell seeding concentration is of major importance for organogenesis in 3D culture, organoid printing allows the user to standardize the process of organoid formation and achieve more reliable and reproducible results.

Micronanotechnology-based multielectrode ar- rays have led to remarkable progress in the field of transmembrane voltage recording of excitable cells. However, providing long-term optoporation- or electroporation-free intracellular access remains a considerable challenge. In this study, a novel type of nanopatterned volcano-shaped micro- electrode (nanovolcano) is described that spontaneously fuses with the cell membrane and permits stable intracellular access. The complex nanostructure was manufactured following a simple and scalable fabrication process based on ion beam etching redeposition. The resulting ring-shaped structure provided passive intracellular access to neonatal rat cardiomyocytes. Intracellular action potentials were successfully recorded in vitro from different devices, and continuous recording for more than 1 h was achieved. By reporting transmembrane action potentials at potentially high spatial resolution without the need to apply physical triggers, the nanovolcanoes show distinct advantages over multielectrode arrays for the assessment of electrophysiological characteristics of cardiomyocyte networks at the transmembrane voltage level over time.

Nanofabrication: 3D multimaterial nanostructures manufactured by ion beam etching redeposition The local sputtering of photoresist sidewalls during ion beam etching enables nanoscale structures to be fabricated. Top-down manufacturing is a scalable approach for the synthesis of nanoscale structures with a range of promising functional properties, and there is an ever growing need to expand the diversity of structures that can be achieved. A team from Ecole Polytechnique Fédérale de Lausanne led by Philippe Renaud now demonstrates how sidewall photoresists might be used to grow structures from a substrate. During ion beam etching of a silicon substrate, the etched material is partly redeposited on the photoresist sidewall. By stripping out the photoresist, sub-100 nm features are achieved, including those composed of multiple materials via the deposition of different materials on the substrate prior to etching.

Cryogels are macroporous materials that display remarkable properties such as high pore inter- connection, large surface to volume ratio and high mechanical stability. As a result they are used in many areas of science spanning from blood separation and filtering to biological scaffolds for cells. However, shaping such material remains challenging because of the harsh conditions of synthesis at temperatures as low as −80◦C. In this paper we propose a solution for the 3D print- ing of cryogels. A micro-fabricated dispensing tool is presented and allows the last-second mixing of the different components for the cryogel synthesis as well as the control in temperature of the material during printing. This dispensing tool is mounted on a 3D printer allowing the multi-layer printing of the cryogel. By controlling the temperature of the dispensing tool during the printing process, we show the possibility to actively and locally tune the pore size of the cryogel. The printed gel are then coated with collagen and seeded with cells. The change in pore size allowed selective cell colonization opening the door to cell patterning through pore size manipulation.

In this paper a nanofluidic molecular filtration system based on soft alginate hydrogel fillings and a solid-state alumina support membrane is presented. The electrostatically controlled diffusion is characterized by partition coefficient of the hydrogel and the flux through the composite membrane for positively and negatively charged dye molecules. The partition coefficient of negatively charged fluorescein sodium molecules into the gel is 2 orders of magnitude lower in 1 mM KCl solution than that in 1 M KCl solution. The molecular transport properties through the hydrogel loaded alumina membrane are solely dominated by the soft nanoporous hydrogel. Such a composite membrane with alginate hydrogel of only 6 wt% shows a selectivity of 5 for the separation of bovine serum albumin (BSA) and bovine hemoglobin (BHb) in high ionic strength solution of phosphate-buffered saline (PBS). (C) 2014 Elsevier B.V. All rights reserved.

Ligament balance is an important and subjective task performed during total knee arthroplasty (TKA) procedure. For this reason, it is desirable to develop instruments to quantitatively assess the soft-tissue balance since excessive imbalance can accelerate prosthesis wear and lead to early surgical revision. The instrumented distractor proposed in this study can assist surgeons on performing ligament balance by measuring the distraction gap and applied load. Also the device allows the determination of the ligament stiffness which can contribute a better understanding of the intrinsic mechanical behavior of the knee joint. Instrumentation of the device involved the use of hall-sensors for measuring the distractor displacement and strain gauges to transduce the force. The sensors were calibrated and tested to demonstrate their suitability for surgical use. Results show the distraction gap can be measured reliably with 0.1 mm accuracy and the distractive loads could be assessed with an accuracy in the range of 4 N. These characteristics are consistent with those have been proposed, in this work, for a device that could assist on performing ligament balance while permitting surgeons evaluation based on his experience. Preliminary results from in vitro tests were in accordance with expected stiffness values for medial collateral ligament (MCL) and lateral collateral ligament (LCL).

Microfluidic platform for the synthesis of complex nanocapsules is presented via a controlled self-assembly. The monodisperse nanocapsules in the range of 50-200 nm consist of a dendritic polyethylene core and a Pluronic copolymer shell. The resultant nanocarriers encapsulate large amount of hydrophobic anticancer drug like paclitaxel while providing a low complement activation as well as sustained release profile with high tunability.

The use of solvent-free microfluidics to fine-tune the physical and chemical properties of chitosan nanoparticles for drug delivery is demonstrated. Nanoparticle self-assembly is driven by pH changes in a water environment, which increases biocompatibility by avoiding organic solvent contamination common with traditional techniques. Controlling the time of mixing (2.5–75 ms) during nanoparticle self-assembly enables us to adjust nanoparticle size and surface potential in order to maximize cellular uptake, which in turn dramatically increases drug effectiveness. The compact nanostructure of these nanoparticles preserves drug potency better than previous nanoparticles, and is more stable during long-term circulation at physiological pH. However, when the nanoparticles encounter a tumor cell and the associated drop in pH, the drug contents are released. Moreover, the loading efficiency of hydrophobic drugs into the nanoparticles increases significantly from previous work to over 95%. The microfluidic techniques used here have applications not just for drug-carrying nanoparticle fabrication, but also for the better control of virtually any self-assembly process.

This paper describes the development of a polyimide-based MEMS strain-sensing device. Finite element analysis was used to investigate an artificial knee implant and assist on device design and to optimize sensing characteristics. The sensing element of the device was fabricated using polyimide micromachining with embedded thin-metallic wires and placed into a knee prosthesis. The device was evaluated experimentally in a mechanical knee simulator using static and dynamic axial load conditions similar to those encountered in vivo. Results indicates the sensor is capable of measuring the strain associated to the total axial forces in the range of approximately 4 times body weight with a good sensitivity and accuracy for events happening within 1 s time window.

Direct collection of extracellular fluid (ECF) plays a central role in the monitoring of neurological disorders. Current approaches using microdialysis catheters are however drastically limited in term of temporal resolution. Here we show a functional in vivo validation of a droplet collection system included at the tip of a neural probe. The system comprises an advanced droplet formation mechanism which enables the collection of neurochemicals present in the brain ECF at high-temporal resolution. The probe was implanted in a rat brain and could successfully collect fluid samples organized in a train of droplets. A microfabricated target plate compatible with most of the surface-based detection methods was specifically developed for sample analysis. The time-resolved brain-fluid samples are analyzed using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The results provide a time evolution picture of the cerebral tissues neurochemical composition for selected elements known for their involvement in neurodegenerative diseases., Current approaches studying the collection of extracellular fluid to monitor neurological disorders have temporal resolution limitations. Here the authors show functional in vivo validation of a droplet collection system included at the tip of a neural probe.

At nanoscale length scales, the properties of particles change rapidly with the slightest change in dimension. The use of a microfluidic platform enables precise control of sub-100 nm organic nanoparticles (NPs) based on polybenzimidazole. Using hydrodynamic flow focusing, we can control the size and shape of the NPs, which in turn controls a number of particle material properties. The anhydrous proton-conducting nature of the prepared NPs allowed us to make a high-performance ion exchange membrane for fuel cell applications, and microfluidic tuning of the NPs allowed us subsequently to tune the fuel cell performance.

This paper describes the development of a polyimide/SU-8 catheter-tip MEMS gauge pressure sensor. Finite element analysis was used to investigate critical parameters, impacting on the device design and sensing characteristics. The sensing element of the device was fabricated by polyimide-based micromachining on a flexible membrane, using embedded thin-film metallic wires as piezoresistive elements. A chamber containing this flexible membrane was sealed using an adapted SU-8 bonding technique. The device was evaluated experimentally and its overall performance compared with a commercial silicon-based pressure sensor. Furthermore, the device use was demonstrated by measuring blood pressure and heart rate in vivo.

This paper describes F-based dry etching and resulting surface properties of biocompatible silicone elastomer. The etch rate of polysiloxane and surface morphology was found to be highly temperature dependent. An increase in temperature results in a significantly higher etch rate and a lower surface roughness. Possible mechanisms of the etching process and the roughness formation on an elastomer surface are discussed. The polysiloxane surface was proved to have hydrophobic characteristics both prior to and after plasma exposure. The results of the preliminary cytotoxicity study are very promising: cell viability on a raw and plasma treated polysiloxane was found to be very high and comparable to control. Due to the acceptable etch rate and absence of toxic contaminations, a F-containing plasma is considered an excellent method for microprocessing of silicone elastomers intended for biomedical applications.

We propose a novel neural probe which combines microfluidic channels with recording and stimulation electrodes. The developed microfabrication approach enables the concentration of every active element such as electrodes and the sampling inlet in close proximity on the same surface. As a first approach, full functional validation is presented in this work (in vivo testing will be presented in the next study). Electrical characterization by impedance spectroscopy is performed in order to assess the electrode properties. An advanced experimental setup enabling the validation of the fluidic functions of the neural probe is also presented. It allowed the achievement of a high temporal resolution (170 ms) during sampling as a result of the integration of a T-junction droplet generator inside the probe. The droplets reached a volume of 0.84 nL and are separated by a non-aqueous phase (perfluoromethyldecalin, PFD). This probe represents an innovative tool for neuroscientists as it can be implanted in precise brain structures while combining electrical stimulation with sampling at a high temporal resolution.

Microstereolithography is a 3D microfabrication technology that is fundamentally different from the techniques commonly used in cleanroom environment for the manufacturing of microelectromechanical system (MEMS) components. Most microfabrication techniques evolved from the microelectronics industry and often use silicon wafers as substrate or carrier material, thin films of metals deposited by evaporation or sputtering, thin layers of polymers deposited by spin coating and patterned by photolithography, and chemical and plasma etching to generate various shapes. Microstereolithography is also a microfabrication technique, but it is related to rapid prototyping technologies, and more precisely to stereolithography, a technique patented in 1986, allowing the fabrication of 3D components by layer-by-layer curing of a photopolymerizable resin with an ultraviolet (UV) laser. Microstereolithography is based on a manufacturing principle very similar to the one of stereolithography, but implements process improvements that result in a far better resolution.

Aims: Here we report a one-step approach for reproducible synthesis of finely tuned targeting multifunctional hybrid nanoparticles (HNPs). Materials & methods: A microfluidic-assisted method was employed for controlled nanoprecipitation of bisphosphonate-conjugated poly(D,L-lactide-co-glycolide) chains, while coencapsulating superparamagnetic iron oxide nanoparticles and the anticancer drug Paclitaxel. Results: Smaller and more compact HNPs with narrower size distribution and higher drug loading were obtained at microfluidic rapid mixing regimen compared with the conventional bulk method. The HNPs were shown to have a strong affinity for hydroxyapatite, as demonstrated in vitro bone-binding assay, which was further supported by molecular dynamics simulation results. In vivo proof of concept study verified the prolonged circulation of targeted microfluidic HNPs. Biodistribution as well as noninvasive bioimaging experiments showed high tumor localization and suppression of targeted HNPs to the bone metastatic tumor. Conclusion: The hybrid bone-targeting nanoparticles with adjustable characteristics can be considered as promising nanoplatforms for various theragnostic applications.