Electrowriting of SU-8 Microfibers.

As microfiber-based additive manufacturing (AM) technologies, melt electrowriting (MEW) and solution electrowriting (SEW) have demonstrated efficacy with more biomedically relevant materials. By processing SU-8 resin using MEW and SEW techniques, a material with substantially different mechanical, thermal, and optical properties than that typically processed is introduced. SU-8 polymer is temperature sensitive and requires the devising of a specific heating protocol to be properly processed. Smooth-surfaced microfibers resulted from MEW of SU8 for a short period (from 30 to 90 min), which provides the greatest control and, thus, reproducibility of the printed microfibers. This investigation explores various parameters influencing the electrowriting process, printing conditions, and post-processing to optimize the fabrication of intricate 3D structures. This work demonstrates the controlled generation of straight filaments and complex multi-layered architectures, which were characterized by brightfield, darkfield, and scanning electron microscopy (SEM). This research opens new avenues for the design and development of 3D-printed photonic systems by leveraging the properties of SU-8 after both MEW and SEW processing.

Combining thermal scanning probe lithography and dry etching for grayscale nanopattern amplification.

Grayscale structured surfaces with nanometer-scale features are used in a growing number of applications in optics and fluidics. Thermal scanning probe lithography achieves a lateral resolution below 10 nm and a vertical resolution below 1 nm, but its maximum depth in polymers is limited. Here, we present an innovative combination of nanowriting in thermal resist and plasma dry etching with substrate cooling, which achieves up to 10-fold amplification of polymer nanopatterns into SiO2 without proportionally increasing surface roughness. Sinusoidal nanopatterns in SiO2 with 400 nm pitch and 150 nm depth are fabricated free of shape distortion after dry etching. To exemplify the possible applications of the proposed method, grayscale dielectric nanostructures are used for scalable manufacturing through nanoimprint lithography and for strain nanoengineering of 2D materials. Such a method for aspect ratio amplification and smooth grayscale nanopatterning has the potential to find application in the fabrication of photonic and nanoelectronic devices.

Microfluidic device combining hydrodynamic and dielectrophoretic trapping for the controlled contact between single micro-sized objects and application to adhesion assays.

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 of hydrodynamic traps holding the first type of cell from below against the fluid flow, and a dielectrophoretic system to force the second type of object to remain in contact with the first one. First, the device is validated by performing an adhesion frequency assay between fibroblasts and fibronectin coated beads. Then, a study is conducted on the modification of the cellular environment to match the dielectrophoretic technology requirements without modifying the cell 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 the pair lifetime. This proof-of-concept device lays the foundations for the development of next generation fast cell-cell interaction technologies.

Aperture-Controlled Fabrication of All-Dielectric Structural Color Pixels.

While interference colors have been known for a long time, conventional color filters have large spatial dimensions and cannot be used to create compact pixelized color pictures. Here we report a simple yet elegant interference-based method of creating microscopic structural color pixels using a single-mask process using standard UV photolithography on an all-dielectric substrate. The technology makes use of the varied aperture-controlled physical deposition rate of low-temperature silicon dioxide inside a hollow cavity to create a thin-film stack with the controlled bottom layer thickness. The stack defines which wavelengths of the reflected light interfere constructively, and thus the cavities act as micrometer-scale pixels of a predefined color. Combinations of such pixels produce vibrant colorful pictures visible to the naked eye. Being fully CMOS-compatible, wafer-scale, and not requiring costly electron-beam lithography, such a method paves the way toward large scale applications of structural colors in commercial products.

Impedance spectroscopy of the cell/nanovolcano interface enables optimization for electrophysiology.

Volcano-shaped microelectrodes have demonstrated superior performance in measuring attenuated intracellular action potentials from cardiomyocyte cultures. However, their application to neuronal cultures has not yet yielded reliable intracellular access. This common pitfall supports a growing consensus in the field that nanostructures need to be pitched to the cell of interest to enable intracellular access. Accordingly, we present a new methodology that enables us to resolve the cell/probe interface noninvasively through impedance spectroscopy. This method measures changes in the seal resistance of single cells in a scalable manner to predict the quality of electrophysiological recordings. In particular, the impact of chemical functionalization and variation of the probe's geometry can be quantitatively measured. We demonstrate this approach on human embryonic kidney cells and primary rodent neurons. Through systematic optimization, the seal resistance can be increased by as much as 20-fold with chemical functionalization, while different probe geometries demonstrated a lower impact. The method presented is therefore well suited to the study of cell coupling to probes designed for electrophysiology, and it is poised to contribute to elucidate the nature and mechanism of plasma membrane disruption by micro/nanostructures.

Rapid exocytosis kinetics measured by amperometry within volcano microelectrodes.

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 µm 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.

High precision, high throughput generation of droplets containing single cells.

The Poisson limit is a major problem for the isolation of single cells in different single-cell technologies and applications. In droplet-based single-cell assays, a scheme that is increasingly popular, the intrinsic randomness during single-cell encapsulation in droplets requires most of the created droplets to be empty, which has a profound impact on the efficiency and throughput of such techniques, and on the predictability of the combinatory droplet assays. Here we present a simple passive microfluidic system overcoming this limitation with unprecedented efficacy, allowing the generation of single-cell droplets for a wide range of operating conditions, with extremely high throughput (more than 22 000 single-cell loaded droplets per minute) and with an extremely low fault ratio (doublets or empty droplets), applicable to any cells and deformable particles. This versatile technique will shift the paradigm of single-cell encapsulation and will impact single-cell sequencing, rare cell isolation, multicellular/bead studies in immunology or cancer biology, etc.

Dielectrophoretic Traps for Efficient Bead and Cell Trapping and Formation of Aggregates of Controlled Size and Composition.

We present a microfluidic dielectrophoretic-actuated system designed to trap chosen single-cell and form controlled cell aggregates. A novel method is proposed to characterize the efficiency of the dielectrophoretic trapping, considering the flow speed but also the heat generated by the traps as limiting criteria in cell-safe manipulation. Two original designs with different manufacturing processes are experimentally compared. The most efficient design is selected and the cell membrane integrity is monitored by fluorescence imaging to guarantee a safe-cell trapping. Design rules are suggested to adapt the traps to multiple-cells trapping and are experimentally validated as we formed aggregates of controlled size and composition with two different types of cells. We provide hereby a simple manufactured tool allowing the controlled manipulation of particles for the composition of multicellular assemblies.

On-Demand Nanoliter Sampling Probe for the Collection of Brain Fluid.

Continuous fluidic sampling systems allow collection of brain biomarkers in vivo. Here, we propose a new sequential and intermittent sampling paradigm using droplets, called Droplet on Demand (DoD). It is implemented in a microfabricated neural probe and alternates phases of analyte removal from the tissue and phases of equilibration of the concentration in the tissue. 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 accurately defined fluidic sequence with controlled timings, volumes, and flow rates, and correct operation is verified by the embedded electrodes and a flow sensor. 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 min 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. Moreover, this makes quantification of biomarkers in the brain possible within acute experiments of only 20-120 min. DoD provides a complementary tool to continuous microdialysis and push-pull sampling probes. Thus, the advances allowed by DoD will benefit quantitative molecular studies in the brain, i.e., for molecules involved in volume transmission or for protein aggregates that form in neurodegenerative diseases over long periods.

Planar hydrodynamic traps and buried channels for bead and cell trapping and releasing.

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.

In-flow electrochemical detection of chemicals in droplets with pyrolysed photoresist electrodes: application as a module for quantification of microsampled dopamine.

The electrochemical quantification of analytes in droplets of PBS separated by a fluorinated phase was investigated. PDMS-fused silica chips with pyrolysed photoresist electrodes were prepared using a simple fabrication technique and used to analyze droplets in flow. Potentiostatic chronoamperometry provided current readouts consistent with mass transport and the concentration inside the droplets. This paper highlights measurements of dopamine in droplets in T-junction microfluidic chips at unprecedently low concentrations, with a limit of detection of 207 nM and a linear range of 0.21-20 µM, giving results similar to continuous flow electrochemistry and allowing the analysis in the striatal extracellular range (<1 µM). The system was applied to the quick and reliable on-line detection of dopamine concentration steps in droplets collected with a microsampling probe in vitro, demonstrating the usefulness of the electrochemical device as a quantification module for microsampled chemicals in droplets.

Microfluidic-assisted bioprinting of tissues and organoids at high cell concentrations.

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.

Nanovolcano microelectrode arrays: toward long-term on-demand registration of transmembrane action potentials by controlled electroporation.

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.

Microfluidics: A New Layer of Control for Extrusion-Based 3D Printing.

Advances in 3D printing have enabled the use of this technology in a growing number of fields, and have started to spark the interest of biologists. Having the particularity of being cell friendly and allowing multimaterial deposition, extrusion-based 3D printing has been shown to be the method of choice for bioprinting. However as biologically relevant constructs often need to be of high resolution and high complexity, new methods are needed, to provide an improved level of control on the deposited biomaterials. In this paper, we demonstrate how microfluidics can be used to add functions to extrusion 3D printers, which widens their field of application. Micromixers can be added to print heads to perform the last-second mixing of multiple components just before resin dispensing, which can be used for the deposition of new polymeric or composite materials, as well as for bioprinting new materials with tailored properties. The integration of micro-concentrators in the print heads allows a significant increase in cell concentration in bioprinting. The addition of rapid microfluidic switching as well as resolution increase through flow focusing are also demonstrated. Those elementary implementations of microfluidic functions for 3D printing pave the way for more complex applications enabling new prospects in 3D printing.

In vivo neurochemical measurements in cerebral tissues using a droplet-based monitoring system.

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.

Multimaterial Nanoporous Membranes Shaped through High Aspect-Ratio Sacrificial Silicon Nanostructures.

We present an innovative fabrication method for solid-state nanoporous membranes based on the casting of sacrificial silicon nanostructures. The process allows the individual definition of geometry and placement of each nanopore through e-beam lithography and is compatible with a wide range of materials without the need to adapt the process to the materials used. We demonstrate the fabrication of membranes integrating high aspect-ratio nanopores with critical dimensions as small as 30 nm, 1.2 µm in length, with round or elongated shapes, and made of silicon dioxide or amorphous carbon. The capability to engineer nanoporous membranes made of a variety of materials and with tailored designs will lead to new applications in the field of electrochemical sensing, flow modulation, or the chemical functionalization of nanopores.

Cell-Imprinted Substrates Modulate Differentiation, Redifferentiation, and Transdifferentiation.

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.

Microfluidic Manipulation of Core/Shell Nanoparticles for Oral Delivery of Chemotherapeutics: A New Treatment Approach for Colorectal Cancer.

A microfluidics approach to synthesize core-shell nanocarriers with high pH tunability is described. The sacrificial shell protects the core layer with the drugs and prevents their release in the severe pH conditions of the gastrointestinal tract, while allowing for drug release in the proximity of a tumor. The proposed nanoparticulate drug-delivery system is designed for the oral administration of cancer therapeutics.

Neural probe combining microelectrodes and a droplet-based microdialysis collection system for high temporal resolution sampling.

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.

On-chip synthesis of fine-tuned bone-seeking hybrid nanoparticles.

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.