Magnetic Cell Centrifuge Platform Performance Study with Different Microsieve Pore Geometries.

The detection and analysis of circulating tumor cells (CTCs) plays a crucial role in clinical practice. However, the heterogeneity and rarity of CTCs make their capture and separation from peripheral blood very difficult while maintaining their structural integrity and viability. We previously demonstrated the effectiveness of the Magnetic Cell Centrifuge Platform (MCCP), which combined the magnetic-labeling cell separation mechanism with the size-based method. In this paper, a comparison of the effectiveness of different microsieve pore geometries toward MCCP is demonstrated to improve the yield of the target cell capture. Firstly, models of a trapped cell with rectangular and circular pore geometries are presented to compare the contact force using finite element numerical simulations. The device performance is then evaluated with both constant pressure and constant flow rate experimental conditions. In addition, the efficient isolation of magnetically labeled Hela cells with red fluorescent proteins (target cells) from Hela cells with green fluorescent protein (background cells) is validated. The experimental results show that the circular sieves yield 97% purity of the target cells from the sample with a throughput of up to 2 µL/s and 66-fold sample enrichment. This finding will pave the way for the design of a higher efficient MCCP systems.

Lippmann waveguide spectrometer with enhanced throughput and bandwidth for space and commercial applications.

This article presents an innovative high spectral resolution waveguide spectrometer, from the concept to the prototype demonstration and the test results. The main goal is to build the smallest possible Fourier transform spectrometer (FTS) with state of the art technology. This waveguide FTS takes advantage of a customized pattern of nano-samplers fabricated on the surface of a planar waveguide that allows the increase of the measurement points necessary for increasing the spectral bandwidth of the FTS in a fully static way. The use of a planar waveguide on the other hand allows enhancing the throughput in a waveguide spectrometer compared to the conventional devices made of single-mode waveguides. A prototype is made in silicon oxynitride/silicon dioxide technology and characterized in the visible range. This waveguide spectrometer shows a nominal bandwidth of 256~nm at a central wavelength of 633~nm thanks to a custom pattern of nanodisks providing a µm sampling interval. The implementation of this innovative waveguide FTS for a real-case scenario is explored and further development of such device for the imaging FTS application is discussed.

An ionic liquid based strain sensor for large displacement measurement.

A robust and low cost ionic liquid based strain sensor is fabricated for high strain measurements in biomedical applications (up to 40 % and higher). A tubular 5 mm long silicone microchannel with an inner diameter of 310 µm and an outer diameter of 650 µm is filled with an ionic liquid. Three ionic liquids have been investigated: 1-butyl-1-methylpyrrolidinium bis (trifluoromethylsulfonyl) imide, ethylammonium nitrate and cholinium ethanoate. When the channel is axially stretched, geometrical deformations change the electrical impedance of the liquid channel. The sensors display a linear response and low hysteresis with an average gauge factors of 1.99 for strains up to 40 %. Additionally, to fix the sensor by surgical stitching to soft biological tissue, a sensor with tube clamps consisting of photopatternable SU-8 epoxy-based resin is proposed.

Resorbable scaffold based chronic neural electrode arrays.

We have developed a novel type of neural electrode array for future brain-machine interfaces (BMI) and neural implants requiring high resolution recording and stimulation on the surface of brain lesions or on the cortex. The devices differ on two points from commonly used thin film electrode arrays: first, the thin film backbone of the implant is exceptionally thin (down to 5 microns) and finely patterned into spring-like structures. This increases the flexibility of the electrode array and allows stretching and conforming better to a quasi spherical cavity surface. Second, the thin film backbone of the device is reinforced with a porous layer of resorbable chitosan. This design aims at minimal invasiveness and low mechanical irritation during prolonged use, while the chitosan matrix ensures the implant is stiff enough for practical handling during the implantation procedure and dissolves afterwards. Furthermore, the chitosan adds haemostatic and antiseptic properties to the implant and improves adhesion. In the article, the design and fabrication process are presented. In vitro and long term in vivo test results over a 12 month period are shown. By adopting the use of a resorbable scaffold-like material as main constituent of neural implants, the presented work opens up the possibility of applying tissue engineering techniques to further improve neural implant technology.

Out-of-Plane Soft Lithography for Soft Pneumatic Microactuator Arrays.

Elastic pneumatic actuators are fueling new devices and applications in soft robotics. Actuator miniaturization is critical to enable soft microsystems for applications in microfluidics and micromanipulation. This work proposes a fabrication technique to make out-of-plane bending microactuators entirely by soft lithography. The only bonding step required is to seal the embedded fluidic channels, assuring the structural integrity of the microactuators. The process consists of fabricating two SU8 mold halves using different lithographic layers. Polydimethilsiloxane is poured on the bottom mold, which is subsequently aligned and assembled with the top mold. The process allows for out-of-plane actuators with a diameter of 300 µm and for fabricating arrays of up to 36 actuators that are row addressable. These active micropillars have an aspect ratio of 1:1.5 and, when pressurized at 1 bar, show a bending angle of ∼30°.

Full-electric microfluidic platform to capture, analyze and selectively release single cells.

Current single-cell technologies require large and expensive equipment, limiting their use to specialized labs. In this paper, we present for the first time a microfluidic device which demonstrates a combined method for full-electric cell capturing, analyzing, and selectively releasing with single-cell resolution. All functionalities are experimentally demonstrated on Saccharomyces cerevisiae. Our microfluidic platform consists of traps centered around a pair of individually accessible coplanar electrodes, positioned under a microfluidic channel. Using this device, we validate our novel Two-Voltage method for trapping single cells by positive dielectrophoresis (pDEP). Cells are attracted to the trap when a high voltage (VH) is applied. A low voltage (VL) holds the already trapped cell in place without attracting additional cells, allowing full control over the number of trapped cells. After trapping, the cells are analyzed by broadband electrochemical impedance spectroscopy. These measurements allow the detection of single cells and the extraction of cell parameters. Additionally, these measurements show a strong correlation between average phase change and cell size, enabling the use of our system for size measurements in biological applications. Finally, our device allows selectively releasing trapped cells by turning off the pDEP signal in their trap. The experimental results show the techniques potential as a full-electric single-cell analysis tool with potential for miniaturization and automation which opens new avenues towards small-scale, high throughput single-cell analysis and sorting lab-on-CMOS devices.

Thin flexible arrays for long-term multi-electrode recordings in macaque primary visual cortex.

Objective.Basic, translational and clinical neuroscience are increasingly focusing on large-scale invasive recordings of neuronal activity. However, in large animals such as nonhuman primates and humans-in which the larger brain size with sulci and gyri imposes additional challenges compared to rodents, there is a huge unmet need to record from hundreds of neurons simultaneously anywhere in the brain for long periods of time. Here, we tested the electrical and mechanical properties of thin, flexible multi-electrode arrays (MEAs) inserted into the primary visual cortex of two macaque monkeys, and assessed their magnetic resonance imaging (MRI) compatibility and their capacity to record extracellular activity over a period of 1 year.Approach.To allow insertion of the floating arrays into the visual cortex, the 20 by 100µm2shafts were temporarily strengthened by means of a resorbable poly(lactic-co-glycolic acid) coating.Main results. After manual insertion of the arrays, theex vivoandin vivoMRI compatibility of the arrays proved to be excellent. We recorded clear single-unit activity from up to 50% of the electrodes, and multi-unit activity (MUA) on 60%-100% of the electrodes, which allowed detailed measurements of the receptive fields and the orientation selectivity of the neurons. Even 1 year after insertion, we obtained significant MUA responses on 70%-100% of the electrodes, while the receptive fields remained remarkably stable over the entire recording period.Significance.Thus, the thin and flexible MEAs we tested offer several crucial advantages compared to existing arrays, most notably in terms of brain tissue compliance, scalability, and brain coverage. Future brain-machine interface applications in humans may strongly benefit from this new generation of chronically implanted MEAs.

A chip-based 128-channel potentiostat for high-throughput studies of bioelectrochemical systems: Optimal electrode potentials for anodic biofilms.

The presence of microorganisms performing extracellular electron transfer has been established in many environments. Research to determine their role is moving slowly due to the high cost of potentiostats and the variance of data with small number of replicates. Here, we present a 128-channel potentiostat, connected to a 128 gold electrode array. Whereas the system is able to perform simultaneously 128 (bio)electrochemical measurements with an independent electrical signal input, the present manufacturing of the array limited the number of effective channels for this study to 77. We assessed the impact of 11 electrode potentials ranging from -0.45V to +0.2V vs. Ag/AgCl (7 replicates per potential) on the growth and electrochemical characteristics of anodic electroactive biofilms (EABs) formed by acetate-fed microbial communities. After 7 days of growth, maximum current was reached for electrodes poised at -0.3V, closely followed by -0.25V and -0.1V to +0.1V, a range well-fitting the midpoint potential of minerals naturally reduced by electroactive bacteria such as Geobacter Sulfurreducens. There was no significant difference in apparent midpoint potential of the EABs (-0.35V), suggesting that the mechanism of heterogeneous electron transfer was not affected by the electrode potential. The EABs poised below current plateau potential (≤-0.3V) exhibited slower growth but higher charge transfer parameters. The high-throughput and high reproducibility provided by the array may have a major facilitating impact on the field of electromicrobiology. Key aspects to improve are data processing algorithms to deal with the vast amount of generated data, and manufacturing of the electrode array itself.

Patterned dextran ester films as a tailorable cell culture platform.

The elucidation of cell-surface interactions and the development of model platforms to help uncover their underlying mechanisms remains vital to the design of effective biomaterials. To this end, dextran palmitates with varying degrees of substitution were synthesised with a multipurpose functionality: an ability to modulate surface energy through surface chemistry, and an ideal thermal behaviour for patterning. Herein, dextran palmitate films are produced by spin coating, and patterned by thermal nanoimprint lithography with nano-to-microscale topographies. These films of moderately hydrophobic polysaccharide esters with low nanoscale roughness performed as well as fibronectin coatings in the culture of bovine aortic endothelial cells. Upon patterning, they display distinct regions of roughness, restricting cell adhesion to the smoothest surfaces, while guiding multicellular arrangements in the patterned topographies. The development of biomaterial interfaces through topochemical fabrication such as this could prove useful in understanding protein and cell-surface interactions.

System for recording from multiple flexible polyimide neural probes in freely behaving animals.

OBJECTIVE: Long-term electrophysiological recordings of neural activity in freely behaving animals are indispensable to advance the understanding of complex brain function. It is a technical challenge to chronically monitor the detailed activity across multiple distributed brain regions in freely behaving animals over a period of months. Here we present a new implant for inserting multiple flexible polyimide probes into freely behaving rats for monitoring the brain activity over a long time period. APPROACH: This brain implant integrates multiple flexible probes in small micromanipulator devices that ensure free behaviour of the animal. The probes are micromachined and the positioning mechanism is 3D-printed using stereolithography. Each probe is lowered by a screw-driven shuttle and guided through an exit tip before penetrating the rat's brain. MAIN RESULTS: The brain implant consists of 16 individually lowerable flexible polyimide probes that contain 16 embedded electrodes adding up to a total of 256 recording channels. The total travel distance is 8 mm. The assembly time of the device was only one day. The electrode impedance values had a mean of 335 kΩ and sample standard deviation of 107 kΩ after gold plating, excluding outliers. SIGNIFICANCE: For the first time, hyperdrive-assisted insertion of flexible multichannel probes was demonstrated. Local field potentials and neuronal spiking activity from freely behaving rats were recorded over months.

Robust gene expression programs underlie recurrent cell states and phenotype switching in melanoma.

Melanoma cells can switch between a melanocytic and a mesenchymal-like state. Scattered evidence indicates that additional intermediate state(s) may exist. Here, to search for such states and decipher their underlying gene regulatory network (GRN), we studied 10 melanoma cultures using single-cell RNA sequencing (RNA-seq) as well as 26 additional cultures using bulk RNA-seq. Although each culture exhibited a unique transcriptome, we identified shared GRNs that underlie the extreme melanocytic and mesenchymal states and the intermediate state. This intermediate state is corroborated by a distinct chromatin landscape and is governed by the transcription factors SOX6, NFATC2, EGR3, ELF1 and ETV4. Single-cell migration assays confirmed the intermediate migratory phenotype of this state. Using time-series sampling of single cells after knockdown of SOX10, we unravelled the sequential and recurrent arrangement of GRNs during phenotype switching. Taken together, these analyses indicate that an intermediate state exists and is driven by a distinct and stable 'mixed' GRN rather than being a symbiotic heterogeneous mix of cells.

Dextran as a Resorbable Coating Material for Flexible Neural Probes.

In the quest for chronically reliable and bio-tolerable brain interfaces there has been a steady evolution towards the use of highly flexible, polymer-based electrode arrays. The reduced mechanical mismatch between implant and brain tissue has shown to reduce the evoked immune response, which in turn has a positive effect on signal stability and noise. Unfortunately, the low stiffness of the implants also has practical repercussions, making surgical insertion extremely difficult. In this work we explore the use of dextran as a coating material that temporarily stiffens the implant, preventing buckling during insertion. The mechanical properties of dextran coated neural probes are characterized, as well as the different parameters which influence the dissolution rate. Tuning parameters, such as coating thickness and molecular weight of the used dextran, allows customization of the stiffness and dissolution time to precisely match the user's needs. Finally, the immunological response to the coated electrodes was analyzed by performing a histological examination after four months of in vivo testing. The results indicated that a very limited amount of glial scar tissue was formed. Neurons have also infiltrated the area that was initially occupied by the dissolving dextran coating. There was no noticeable drop in neuron density around the site of implantation, confirming the suitability of the coating as a temporary aid during implantation of highly flexible polymer-based neural probes.

A foldable electrode array for 3D recording of deep-seated abnormal brain cavities.

OBJECTIVE: This study describes the design and microfabrication of a foldable thin-film neural implant and investigates its suitability for electrical recording of deep-lying brain cavity walls. APPROACH: A new type of foldable neural electrode array is presented, which can be inserted through a cannula. The microfabricated electrode is specifically designed for electrical recording of the cavity wall of thalamic lesions resulting from stroke. The proof-of-concept is demonstrated by measurements in rat brain cavities. On implantation, the electrode array unfolds in the brain cavity, contacting the cavity walls and allowing recording at multiple anatomical locations. A three-layer microfabrication process based on UV-lithography and Reactive Ion Etching is described. Electrochemical characterization of the electrode is performed in addition to an in vivo experiment in which the implantation procedure and the unfolding of the electrode are tested and visualized. MAIN RESULTS: Electrochemical characterization validated the suitability of the electrode for in vivo use. CT imaging confirmed the unfolding of the electrode in the brain cavity and analysis of recorded local field potentials showed the ability to record neural signals of biological origin. SIGNIFICANCE: The conducted research confirms that it is possible to record neural activity from the inside wall of brain cavities at various anatomical locations after a single implantation procedure. This opens up possibilities towards research of abnormal brain cavities and the clinical conditions associated with them, such as central post-stroke pain.

Extracellular matrix proteins as temporary coating for thin-film neural implants.

OBJECTIVE: This study investigates the suitability of a thin sheet of extracellular matrix (ECM) proteins as a resorbable coating for temporarily reinforcing fragile or ultra-low stiffness thin-film neural implants to be placed on the brain, i.e. microelectrocorticographic (µECOG) implants. APPROACH: Thin-film polyimide-based electrode arrays were fabricated using lithographic methods. ECM was harvested from porcine tissue by a decellularization method and coated around the arrays. Mechanical tests and an in vivo experiment on rats were conducted, followed by a histological tissue study combined with a statistical equivalence test (confidence interval approach, 0.05 significance level) to compare the test group with an uncoated control group. MAIN RESULTS: After 3 months, no significant damage was found based on GFAP and NeuN staining of the relevant brain areas. SIGNIFICANCE: The study shows that ECM sheets are a suitable temporary coating for thin µECOG neural implants.

Insulation lifetime improvement of polyimide thin film neural implants.

OBJECTIVE: This work deals with studying and improving the insulation lifetime of polyimide-insulated thin film neural implants, or related polyimide-based medical implants. APPROACH: The evolution of the leak impedance of insulated conductors was investigated in saline water at 40 °C. The fabrication process as commonly found in literature for polyimide/platinum/polyimide microelectrode arrays was compared with three possible improvements: one based on lowering the curing temperature of the lower layer, one based on chemical activation and one based on an additional plasma activation step. MAIN RESULTS: The lower curing temperature process was found to yield a 7.5-fold improved lifetime compared with the state of the art process. Also, the leak impedances found after soak testing are an order of magnitude higher compared to the standard process. SIGNIFICANCE: By improving the lifetime and insulation impedance of polyimide insulation with one order of magnitude, this work increases the applicability of polyimide in chronic thin film neural implants considerably.

Digital microfluidics-enabled single-molecule detection by printing and sealing single magnetic beads in femtoliter droplets.

Digital microfluidics is introduced as a novel platform with unique advantages for performing single-molecule detection. We demonstrate how superparamagnetic beads, used for capturing single protein molecules, can be printed with unprecedentedly high loading efficiency and single bead resolution on an electrowetting-on-dielectric-based digital microfluidic chip by micropatterning the Teflon-AF surface of the device. By transporting droplets containing suspended superparamagnetic beads over a hydrophilic-in-hydrophobic micropatterned Teflon-AF surface, single beads are trapped inside the hydrophilic microwells due to their selective wettability and tailored dimensions. Digital microfluidics presents the following advantages for printing and sealing magnetic beads for single-molecule detection: (i) droplets containing suspended beads can be transported back and forth over the array of hydrophilic microwells to obtain high loading efficiencies of microwells with single beads, (ii) the use of hydrophilic-in-hydrophobic patterns permits the use of a magnet to speed up the bead transfer process to the wells, while the receding droplet meniscus removes excess beads off the chip surface and thereby shortens the bead patterning time, and (iii) reagents can be transported over the printed beads multiple times, while capillary forces and a magnet hold the printed beads in place. High loading efficiencies (98% with a CV of 0.9%) of single beads in microwells were obtained by transporting droplets of suspended beads over the array 10 times in less than 1 min, which is much higher than previously reported methods (40-60%), while the total surface area needed for performing single-molecule detection can be decreased. The performance of the device was demonstrated by fluorescent detection of the presence of the biotinylated enzyme ß-galactosidase on streptavidin-coated beads with a linear dynamic range of 4 orders of magnitude ranging from 10 aM to 90 fM.

Biofunctionalization of electrowetting-on-dielectric digital microfluidic chips for miniaturized cell-based applications.

In this paper we report on the controlled biofunctionalization of the hydrophobic layer of electrowetting-on-dielectric (EWOD) based microfluidic chips with the aim to execute (adherent) cell-based assays. The biofunctionalization technique involves a dry lift-off method with an easy to remove Parylene-C mask and allows the creation of spatially controlled micropatches of biomolecules in the Teflon-AF(®) layer of the chip. Compared to conventional methods, this method (i) is fully biocompatible; and (ii) leaves the hydrophobicity of the chip surface unaffected by the fabrication process, which is a crucial feature for digital microfluidic chips. In addition, full control of the geometry and the dimensions of the micropatches is achieved, allowing cells to be arrayed as cell clusters or as single cells on the digital microfluidic chip surface. The dry Parylene-C lift-off technique proves to have great potential for precise biofunctionalization of digital microfluidic chips, and can enhance their use for heterogeneous bio-assays that are of interest in various biomedical applications.