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Summary Duchenne muscular dystrophy (DMD) is a rare X-linked recessive disease that is associated with severe progressive muscle degeneration culminating in death due to cardiorespiratory failure. We previously observed an unexpected proliferation-independent telomere shortening in cardiomyocytes of a DMD mouse model. Here, we provide mechanistic insights using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using traction force microscopy, we show that DMD hiPSC-CMs exhibit deficits in force generation on fibrotic-like bioengineered hydrogels, aberrant calcium handling, and increased reactive oxygen species levels. Furthermore, we observed a progressive post-mitotic telomere shortening in DMD hiPSC-CMs coincident with downregulation of shelterin complex, telomere capping proteins, and activation of the p53 DNA damage response. This telomere shortening is blocked by blebbistatin, which inhibits contraction in DMD cardiomyocytes. Our studies underscore the role of fibrotic stiffening in the etiology of DMD cardiomyopathy. In addition, our data indicate that telomere shortening is progressive, contraction dependent, and mechanosensitive, and suggest points of therapeutic intervention., Highlights • DMD hiPSC-CMs exhibit aberrant calcium handling and defective force generation • DMD hiPSC-CMs undergo proliferation-independent telomere shortening • Telomere shortening activates the p53 DNA damage pathway • Telomere shortening in DMD hiPSC-CMs is contraction dependent, In this article, Chang, Blau, and colleagues show that Duchenne muscular dystrophy (DMD) iPSC-derived cardiomyocytes exhibit proliferation-independent telomere shortening, p53 activation and mitochondrial dysfunction.

Human-induced pluripotent stem cell-derived cardiomyocytes are a potentially unlimited cell source and promising patient-specific in vitro model of cardiac diseases. Yet, these cells are limited by immaturity and population heterogeneity. Current in vitro studies aiming at better understanding of the mechanical and chemical cues in the microenvironment that drive cellular maturation involve deformable materials and precise manipulation of the microenvironment with, for example, micropatterns. Such microenvironment manipulation most often involves microfabrication protocols which are time-consuming, require cleanroom facilities and photolithography expertise. Here, we present a method to increase the scale of the fabrication pipeline, thereby enabling large-batch generation of shelf-stable microenvironment protein templates on glass chips. This decreases fabrication time and allows for more flexibility in the subsequent steps, for example, in tuning the material properties and the selection of extracellular matrix or cell proteins. Further, the fabrication of deformable hydrogels has been optimized for compatibility with these templates, in addition to the templates being able to be used to acquire protein patterns directly on the glass chips. With our approach, we have successfully controlled the shapes of cardiomyocytes seeded on Matrigel-patterned hydrogels. Additional fundersThe National Science Foundation (CMMI 1834760). E.A.C. was supported by the National Science Foundation Graduate Research Fellowship (DGE-1656518) and the Ford Foundation Pre-doctoral Fellowship. K.V.L. was supported by the National Science Foundation Graduate Research Fellowship (DGE-1650114). G.V.T. was supported by the Data Driven Biology National Science Foundation Research Traineeship (DGE-2125644).

The study of human cardiomyopathies and the development and testing of new therapies has long been limited by the availability of appropriate in vitro model systems. Cardiomyocytes are highly specialized cells whose internal structure and contractile function are sensitive to the local microenvironment and the combination of mechanical and biochemical cues they receive. The complementary technologies of human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CMs) and microphysiological systems (MPS) allow for precise control of the genetics and microenvironment of human cells in in vitro contexts. These combined systems also enable quantitative measurement of mechanical function and intracellular organization. This review describes relevant factors in the myocardium microenvironment that affect CM structure and mechanical function and demonstrates the application of several engineered microphysiological systems for studying development, disease, and drug discovery.

Significance Heart disease is the leading cause of death worldwide, and hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, affecting over 1 in 200 people. Mutations in myosin, the motor protein responsible for contraction of the heart, are a common cause of HCM but have diverse effects on the biomechanics of the myosin protein. We demonstrate that complex biomechanical effects of mutations associated with heart disease can be effectively studied and understood using a multiscale experimental and computational modeling approach. This work confirms an important role for disruption of the super relaxed state for one particular HCM mutation and provides an approach that can be extended to aid in the understanding of disease mechanisms for different mutations., Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1,000 mutations, many in β-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned human induced pluripotent derived stem cell (hiPSC)–cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are powerfulin-vitromodels to study the mechanisms underlying cardiomyopathies and cardiotoxicity. To understand how cellular mechanisms affect the heart, it is crucial to quantify the contractile function in single hiPSC-CMs over time, however, such measurements remain demanding and low-throughput, and are too seldom considered.We developed an open-access, versatile, streamlined, and highly automated pipeline to address these challenges and enable quantitativetrackingof thecontractiledynamics of single hiPSC- CMs over time:ConTraX. Three interlocking software modules enable: (i) parameter-based localization and selection of single hiPSC-CMs; (ii) automated video acquisition of >200 cells/hour; and (iii) streamlined measurements of the contractile parameters via traction force microscopy. UsingConTraX, we analyzed >2,753 hiPSC-CMs over time under orthogonal experimental conditions in terms of culture media and substrate stiffnesses. Using undirected high-dimensional clustering, we dissected the complex diversity of contractile phenotypes in hiPSC-CM populations and revealed converging maturation patterns.Our modularConTraXpipeline empowers biologists with a potent quantitative analytic tool applicable to the development of cardiac therapies.

Duchenne muscular dystrophy (DMD) is a progressive genetic myopathy that leads to heart failure from dilated cardiomyopathy by early adulthood. Recent evidence suggests that tamoxifen, a selective estrogen receptor modulator widely used to treat breast cancer, ameliorates DMD cardiomyopathy. However, the mechanism of action of 4-hydroxytamoxifen, the active metabolite of tamoxifen, on cardiomyocyte function remains unclear. To examine the effects of chronic 4-hydroxytamoxifen treatment, we used state-of-the-art human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and a bioengineered platform to model DMD. We assessed the beating rate and beating velocity of iPSC-CMs in monolayers and as single cells on micropatterns that promote a physiological cardiomyocyte morphology. We found that 4-hydroxytamoxifen treatment of DMD iPSC-CMs decreased beating rate, increased beating velocity, and ameliorated calcium-handling deficits, leading to prolonged viability. Our study highlights the utility of a bioengineered iPSC-CM platform for drug testing and underscores the potential of repurposing tamoxifen as a therapy for DMD cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1000 mutations, many in β-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super-relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned hiPSC-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super-relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.Significance StatementHeart disease is the leading cause of death worldwide, and hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, affecting over 1 in 200 people. Mutations in myosin, the motor protein responsible for contraction of the heart, are a common cause of HCM but have diverse effects on the biomechanics of the myosin protein. We demonstrate that complex biomechanical effects of mutations associated with heart disease can be effectively studied and understood using a multi-scale experimental and computational modeling approach. This work confirmed an important role for disruption of the super-relaxed state for one particular HCM mutation, and our approach can be extended to aid in the development of new targeted therapies for patients with different mutations.

Traction Force Microscopy (TFM) has become a well-established technique to assay the biophysical force produced by cells cultured on soft substrates of controlled stiffness. However, experimental conditions as well as computational implementations can have a large impact on the analysis results accuracy and reproducibility. While this can be alleviated using appropriate controls and a rigorous analytical approach, the comparison of results across studies remains difficult and there is a need for validation and benchmarking tools. To validate the accuracy of and compare various computational TFM analysis algorithms, we developed a virtualin silicomodel of a cell contracting on a soft substrate of controlled stiffness. The model utilizes user-defined parameters for the cell dimensions as well as for the strength and spatial distribution of a contraction dipole to calculate the deformation that would result on a soft substrate due to the cell contraction. The deformation is computed using the forward analytical stress-strain tensor calculation in the Fourier space. The resulting displacement field is used to apply, using image processing, a deformation on a real or simulated image of fluorescent microspheres embedded into a soft hydrogel, which is normally obtain experimentally by TFM imaging. The deformation field and resulting image then serve as input in the PIV and TFM analysis. The model also enables to create movies of a dynamic cell contraction, such as that of a cardiomyocyte, to validate the time accuracy of the TFM analysis after application of image processing algorithm, such as denoising. Our tool therefore addresses the need for validation and standardization of TFM analytical algorithms and its experimental implementations.

Duchenne Muscular Dystrophy (DMD) is a lethal X-chromosome linked disease that affects ~1:3500 boys and culminates in heart failure in early adulthood. DMD results from >200 possible dystrophin mutations. The lack of dystrophin disrupts the anchoring of the sarcomere to the extracellular matrix (ECM), impairing cardiomyocyte function and cardiac contraction. With disease progression, the heart tissue stiffness increases as fibrosis progresses and a dilated cardiomyopathy phenotype develops. We hypothesize that disease progression accelerates because of a positive feedback loop involving fibrotic stiffening and mechanosensing. Our preliminary results show that single human-derived pluripotent stem cell (hiPSC) cardiomyocytes (CMs) with DMD mutations have a dramatically reduced ability to produce force compared to isogenic controls on stiffer substrates mimicking a fibrotic state, but not on soft healthy substrates. This stiffness-dependent contractile deficiency correlates with an increase in reactive oxygen species (ROS) and mitochondrial dysfunction, as well as other markers of cellular stress response and senescence. Here, we ask how stiffness affects the progression of DMD cardiomyopathy and aim to draw a link between the lack of dystrophin and force production deficiency in hiPSC-CMs with or without DMD mutations in an isogenic background. Using a novel assay and algorithm, we simultaneously measure extracellular force production by traction force microscopy (TFM) and single sarcomere dynamics (SSD) in single hiPSC-CMs adhered onto ECM micropatterns on top of a hydrogel of stiffness matching that of healthy (~10kPa) or fibrotic (~35kPa) tissue. We induce the single hiPSC-CMs to adopt a physiologic elongated 1:7 aspect ratio using microcontact printing of ECM protein. Using this platform, we determine how the lack of dystrophin impacts sarcomere architecture, sarcomere contractility, and force generation in the etiology of DMD cardiac disease.

Electrostatic sampling is a promising method for the collection of bioaerosol particles. Although the underlying physics responsible for particle collection are well understood, the collection efficiency of simple passive electrostatic samplers is difficult to predict. Under these conditions, the collection efficiency becomes very sensitive to ambient air current and particle size, especially for submicron particles relevant for airborne virus transmission. In this paper, we compare two electrostatic aerosol sampler designs, a commercial product consisting of a flat collector plate located in the same plane as the charging needles and an axisymmetric design sampling directly to a liquid droplet. The aerosol particle collection efficiency of the samplers is investigated for particle size ranging from 0.25 to 2 µm while the air flow velocity surrounding the samplers is varied from 0.3 to 1 m s–1. For the planar design, at all ambient flow velocities, the submicron fraction of the particles captured originates in streamlines up to a maximum of 75 mm above the surface of the device collector, which greatly limits the volume of air being effectively sampled. The axisymmetric design features a non-monotonic capture efficiency as a function of particle size, with a minimum between 0.4 and 0.8 µm. The flow field in the inter-electrode region, captured using particle image velocimetry (PIV) reveals the presence of strong recirculation zones that can be responsible for the increased collection efficiency for very small particles.

Bead-based microwell array technology is growing as an ultrasensitive analysis tool as exemplified by the successful commercial applications from Illumina and Quanterix for nucleic acid analysis and ultrasensitive protein measurements, respectively. High-efficiency seeding of magnetic beads is key for these applications and is enhanced by hydrophilic-in-hydrophobic microwell arrays, which are unfortunately often expensive or labor-intensive to manufacture. Here, we demonstrate a new single-step manufacturing approach for imprinting cheap and disposable hydrophilic-in-hydrophobic microwell arrays suitable for digital bioassays. Imprinting of arrays with hydrophilic-in-hydrophobic microwells is made possible using an innovative surface energy replication approach by means of a hydrophobic thiol-ene polymer formulation. In this polymer, hydrophobic-moiety-containing monomers self-assemble at the hydrophobic surface of the imprinting stamp, which results in a hydrophobic replica surface after polymerization. After removing the stamp, microwells with hydrophobic walls and a hydrophilic bottom are obtained. We demonstrate that the hydrophilic-in-hydrophobic imprinted microwell arrays enable successful and efficient self-assembly of individual water droplets and seeding of magnetic beads with loading efficiencies up to 96%. We also demonstrate the suitability of the microwell arrays for the isolation and digital counting of single molecules achieving a limit of detection of 17.4 aM when performing a streptavidin-biotin binding assay as model system. Since this approach is up-scalable through reaction injection molding, we expect it will contribute substantially to the translation of ultrasensitive digital microwell array technology toward diagnostic applications. ispartof: ACS Applied Materials and Interfaces vol:9 issue:12 pages:10418-10426 ispartof: location:United States status: published

Author(s): Eguchi, Asuka; Chang, Alex C; Pardon, Gaspard; Pruitt, Beth L; Bernstein, Daniel; Blau, Helen M | Abstract: Duchenne muscular dystrophy (DMD) is a devastating X-linked genetic disorder that affects 1 in 3500 males. Characterized by progressive muscle degeneration that culminates in respiratory failure and dilated cardiomyopathy, DMD is caused by a lack of dystrophin, a protein that provides structural support between the sarcomeric cytoskeleton and the extracellular matrix. Loss of dystrophin leads to a leaky plasma membrane, contractile stress, and disruption of cellular homeostasis. However, the molecular mechanism that eventually leads to cell death remains to be explored. Recently, the Blau lab discovered a pathogenic link between DMD cardiomyopathy and telomere dysfunction. While mdx mice that lack functional dystrophin do not exhibit dilated cardiomyopathy as in human patients, when crossed with mTR mice that lack the RNA component of telomerase (TERC), mdx mice with “humanized” telomere lengths fully manifested the severe muscle wasting and cardiac failure seen in patients. Notably, the longer telomeres, characteristic of mice, appear to be cardioprotective. Importantly, we observed telomere shortening in cardiomyocytes, but not other cell types, of DMD patients compared to age-matched controls. Preliminary data suggests that contractile stress due to the lack of dystrophin leads to a pathogenic feed-forward loop which ultimately culminates in cardiomyocyte cell death. Using human iPS cells derived from DMD patients, we have modeled telomere shortening and aspects of cardiomyocyte dysfunction characteristic of DMD, including aberrant calcium transients, mechanical stress, and arrhythmia. By comparing cardiomyocytes derived from DMD iPS cells with those from CRISPR-corrected isogenic controls on patterned bioengineered hydrogel platforms of varying stiffness, we can study the role of fibrotic stiffening in the myocardium in the premature demise of DMD cardiomyocytes. Pinpointing the early molecular events that trigger the pathogenic feed-forward loop will provide strategies for intervention to ameliorate DMD cardiomyopathy.

Duchenne Muscular Dystrophy (DMD) is a X-link disease affecting ~1:3500 boys per year and culminating with heart failure in early adulthood. DMD results from >200 possible genetic mutations on dystrophin. The lack of dystrophin disrupts the anchoring of the cell sarcomere to the extracellular matrix (ECM), affecting the cardiac contraction. With disease progression, in an attempt to mitigate the subcellular defects, the tissue stiffness and ECM composition remodel in association with a dilated cardiomyopathy phenotype and fibrosis. Our hypothesis is that disease progression is accelerating because of this remodelling, through a positive feedback loop involving multiple mechanosensing pathways. Here, we use a single-cell assay platform to model the effect of fibrotic remodelling in DMD. This platform allows measuring the force production of single human-derived pluripotent stem cell (hiPSC) cardiomyocytes (CMs). The substrate stiffness can be controlled to match that of healthy (~10kPa) or fibrotic (~35kPa) tissue. In addition, single iPSC-CMs are patterned in an elongated 1:7 aspect ratio using microcontact printing of ECM protein. This enhances their intracellular structural maturity towards a more mature adult phenotype. This renders our in-vitro model more representative of the human pathology and greatly improves our measurements standardization. Our results show that single iPSC-CMs with DMD mutations have a dramatically reduced ability to produce force on stiffer substrates compared to their isogenic control. This loss of contractile function correlates with an increase in reactive oxygen species (ROS) and mitochondria dysfunction, as well as with other markers of stress response and cellular senescence.We are asking how the remodeling of the ECM stiffness and composition is signaled from the outside-in and affects the progression of the disease phenotype. The further development of our platform and approach will allow for more accurate in-vitro modeling of cardiac diseases and greatly increase our understanding of the underlying biophysics of mechanosensing.

Patterning of micro- and nanoscale topologies and surface properties of polymer devices is of particular importance for a broad range of life science applications, including cell-adhesion assays and highly sensitive bioassays. The manufacturing of such devices necessitates cumbersome multiple-step fabrication procedures and results in surface properties which degrade over time. This critically hinders their wide-spread dissemination. Here, we simultaneously mold and surface energy pattern microstructures in off-stoichiometric thiol-ene by area-selective monomer self-assembly in a rapid micro-reaction injection molding cycle. We replicated arrays of 1,843,650 hydrophilic-in-hydrophobic femtolitre-wells with long-term stable surface properties and magnetically trapped beads with 75% and 87.2% efficiency in single- and multiple-seeding events, respectively. These results form the basis for ultrasensitive digital biosensors, specifically, and for the fabrication of medical devices and life science research tools, generally., Simultaneous microstructuring and surface modification of microfluidic bioreactors Researchers from Sweden and Belgium have developed a simplified technique to create microfluidic devices with patterns of modified surface properties. Microfluidic devices, such as the one demonstrated here by a team led by Wouter van der Wijngaart, from KTH Royal Institute of Technology, Sweden, utilize polymer microstructures with hydrophobic and hydrophilic properties to manipulate fluids on a tiny scale. This enables ‘lab-on-a-chip’ experiments with a diverse array of applications. However, current devices suffer from complicated fabrication processes and a limited lifespan. van der Wijngaart and his team used the polymer ‘off-stoichiometric thiol-ene’ to merge a rapid replication technique called reaction injection molding with a method to self-assemble hydrophobic and hydrophilic micropatterns in a single, rapid fabrication step. The researchers hope that their results will form the basis for future microfluidic chip manufacturing and lead to the creation of ultrasensitive life science research tools.

Author(s): Engel, Leeya; Gaietta, Guido; Dow, Liam; Swift, Mark; Pardon, Gaspard; Volkmann, Niels; Weis, William; Hanein, Dorit; Pruitt, Beth | Abstract: ABSTRACT Cryogenic electron tomography is the highest resolution tool available for structural analysis of macromolecular organization inside cells. Micropatterning of extracellular matrix (ECM) proteins is an established in vitro cell culture technique used to control cell shape. Recent traction force microscopy studies have shown correlation between cell morphology and the regulation of force transmission. However, it remains unknown how cells sustain increased strain energy states and localized stresses at the supramolecular level. Here, we report a technology to enable direct observation of mesoscale organization in epithelial cells under morphological modulation, using a maskless protein photopatterning method to confine cells to ECM micropatterns on electron microscopy substrates. These micropatterned cell culture substrates can be used in mechanobiology research to correlate changes in nanometer-scale organization at cell-cell and cell-ECM contacts to strain energy states and traction stress distribution in the cell.

INTRODUCTION: The orientation of collagen fibers in native tissues plays an important role in cell signaling and mediates the progression of tumor cells in breast cancer by a contact guidance mechanism. Understanding how migration of epithelial cells is directed by the alignment of collagen fibers requires in vitro assays with standardized orientations of collagen fibers. METHODS: To address this issue, we produced micro-stripes with aligned collagen fibers using an easy-to-use and versatile approach based on the aspiration of a collagen solution within a microchannel. Glass coverslips were functionalized with a (3-aminopropyl)triethoxysilane/glutaraldehyde linkage to covalently anchor micro-stripes of aligned collagen fibers, whereas microchannels were functionalized with a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) nonionic triblock polymer to prevent adhesion of the collagen micro-stripes. RESULTS: Using this strategy, microchannels can be peeled off to expose micro-stripes of aligned collagen fibers without affecting their mechanical integrity. We used time-lapse confocal reflection microscopy to characterize the polymerization kinetics of collagen networks for different concentrations and the orientation of collagen fibers as a function of the microchannel width. Our results indicate a non-linear concentration dependence of the area of fluorescence, suggesting that the architecture of collagen networks is sensitive to small changes in concentration. We show the possibility to influence the collagen fibril coverage by adjusting the concentration of the collagen solution. CONCLUSION: We applied this novel approach to study the migration of epithelial cells, demonstrating that collagen micro-stripes with aligned fibers represent a valuable in-vitro assay for studying cell contact guidance mechanisms. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12195-019-00600-4) contains supplementary material, which is available to authorized users.

Although tissue engineering using human-induced pluripotent stem cells is a promising approach for treatment of cardiovascular diseases, some limiting factors include the survival, electrical integration, maturity, scalability, and immune response of three-dimensional (3D) engineered tissues. Here we discuss these important roadblocks facing the tissue engineering field and suggest potential approaches to overcome these challenges., In this Comment, Ngan Huang et al. discuss recent advances in cardiovascular tissue engineering and some of the main challenges that remain in translating these advances to the clinic. The authors propose future direction for the field to focus research efforts.

In this work, the development of a point-of-care (PoC) system to capture aerosol from litres of air directly onto a microfluidic lab-on-chip for subsequent analysis is addressed. The system involves an electrostatic precipitator that uses corona charging and electrophoretic transport to capture aerosol droplets onto a microfluidic air-to-liquid interface for downstream analysis. A theoretical study of the governing geometric and operational parameters for optimal electrostatic precipitation is presented. The fabrication of an electrostatic precipitator prototype and its experimental validation using a laboratory-generated aerosolized dye is described. Collection efficiencies were comparable to those of a state-of-the-art Biosampler impinger, with the significant advantage of providing samples that are at least 10 times more concentrated. Finally, we discuss the potential of such a system for breath-based diagnostics. QC 20150223 Rappid

A new and easy-to-use method that allows for mold-free and rapid prototyping of microfluidic devices, comprising channels, access holes, and surface-modified patterns, is presented. The innovative method is based on direct photolithographic patterning of an off-stoichiometry thiol-ene (OSTE) polymer formulation, tailor-made for photolithography, which offers unprecedented spatial resolution and allows for efficient, robust and reliable, room temperature surface modification and glue-free, covalent room temperature bonding. This mold-free process does not require clean room equipment and therefore allows for rapid, i.e., less than one hour, design-fabricate-test cycles, using a material suited for larger-scale production. The excellent photolithographic properties of this new OSTE formulation allow patterning with unprecedented, for thiol-ene polymer systems, resolution in hundreds of micrometers thick layers, 200 mu m thick in this work. Moreover, we demonstrated robust, covalent and spatially controlled modification of the microchannel surfaces with an initial contact angle of 76A degrees by patterning hydrophobic/hydrophilic areas with contact angles of 102A degrees and 43A degrees, respectively.

Today, despite the growing interest in nanofluidics, the descriptions of the many complex physical phenomena occurring at this scale remain scattered in the literature. Due to the additional complexity encountered when considering electrostatic nanofluidic gating, it is important to regroup several relevant theories and discuss them with regard to this application. In this work, we present a theoretical study of electrostatically gated phenomena and propose a model for the electrostatic gating of ion and molecular transport in nanochannels. In addition to the classical electrokinetic equations, that are reviewed in this work, several relevant phenomena are considered and combined to describe gating effects on nanofluidic properties more accurately. Dynamic surface charging is accounted for and is shown to be an essential element for electrostatic gating. The autoprotolysis of water is also considered to allow for accurate computing of the surface charge. Modifications of the Nernst-Planck equations are considered for more accurate computing of the concentration profiles at higher surface potentials by accounting for ion crowding near charge walls. The sensitivity of several parameters to the electric field and ion crowding is also studied. Each of these models is described separately before their implementation in a finite element model. The model is verified against previous experimental work. Finally, the model is used to simulate the tuning of the ionic current through the nanochannel via electrostatic gating. The influence of the additional models on these results is discussed. Guidelines for potentially better gating efficiencies are finally proposed. QC 20131129 NanoGate

Surface Energy Mimicking : Simultaneous Replication of Hydrophilic and Superhydrophobic Micropatterns through Area-Selective Monomers Self-Assembly (Adv. Mater. Interfaces 17/2016)

The control and permanent modification of the surface properties of polymers is a critical enabler for many applications. Here, we demonstrate a strategy, which we call surface energy mimicking, for the spontaneous replication of micropatterns of surface energies ranging from hydrophilic to superhydrophobic from a mold to several replicas. We introduce surface energy mimicking, enabling spontaneous replication of micropatterns (2D and 2.5D) of different surface energies, and enabled by self-assembly of functional mimicking monomers within a polymer matrix. We demonstrate replication of surface energies ranging from hydrophilic to superhydrophobic, and self-assembly of picoliter-droplet arrays on replicated micropatterned arrays containing hydrophilic patches in a hydrophobic surface. QC 20161024

We report a novel polymer material formulation and stamp-molding technique that enable rapid single-step manufacturing of hydrophilic-in-hydrophobic microwell arrays. We developed a modified thiol-ene-epoxy polymer (mOSTE+) formulation that mimics the surface energy of its mold during polymerization. The polymer inherits the surface energy from the mold through molecular self-assembly, in which functional monomers self-assemble at the interface between the liquid prepolymer and the mold surface. Combining this novel mOSTE+ material with a stamp-molding process leads to simultaneous surface energy mimicking and micro-structuring. This method was used to manufacture microwells with hydrophilic bottom and hydrophobic sidewall, depressed in a surrounding hydrophobic surface. The microwell arrays were successfully tested for the self-assembly of 62∲000 femtoliter-droplets. Such femtoliter droplet arrays are useful for, e.g., digital ELISA and single cell/molecule analysis applications.

We report a novel surface-energy patterning phenomenon, in which a novel polymer composition inherits the surface energy of the medium it is in contact with during polymerization. This surface property mimicking process occurs via spontaneous selective molecular alignment of hydrophilic and hydrophobic monomers mixed into an off-stoichiometry thiol-ene (OSTE) formulation. This single-step method for simultaneous structuring and surface energy micropatterning of polymer structures is potentially more robust and lower cost than state-of-the-art processes requiring post-processing surface modification steps. We further demonstrate the self-assembly of a liquid droplet array on the replicated polymer surfaces. QC 20140521 Rappid

Functional nanoporous materials are promising for a number of applications ranging from selective biofiltration to fuel cell electrodes. This work reports the functionalization of nanoporous membranes using atomic layer deposition (ALD). ALD is used to conformally deposit platinum (Pt) and aluminum oxide (Al2O3) on Pt in nanopores to form a metal-insulator stack inside the nanopore. Deposition of these materials inside nanopores allows the addition of extra functionalities to nanoporous materials such as anodic aluminum oxide (AAO) membranes. Conformal deposition of Pt on such materials enables increased performances for electrochemical sensing applications or fuel cell electrodes. An additional conformal Al2O3 layer on such a Pt film forms a metal-insulator-electrolyte system, enabling field effect control of the nanofluidic properties of the membrane. This opens novel possibilities in electrically controlled biofiltration. In this work, the deposition of these two materials on AAO membranes is investigated theoretically and experimentally. Successful process parameters are proposed for a reliable and cost-effective conformal deposition on high aspect ratio three-dimensional nanostructures. A device consisting of a silicon chip supporting an AAO membrane of 6 mm diameter and 1.3 mu m thickness with 80 nm diameter pores is fabricated. The pore diameter is reduced to 40 nm by a conformal deposition of 11 nm Pt and 9 nm Al2O3 using ALD. QC 20130110 NanoGate

This work presents a predictive and today's most complete model for electrostatic gating of nanofluidic channels. The model is based on the classical mean field equations and accounts for the surface reactivity. The model is successfully verified against previously published experimental data [1]. The fabrication concept of a novel device is introduced and its predicted performances are discussed with the help of the presented model. A ~40x performance improvement, in terms of tunability of the ionic conductivity in the nanochannel, in comparison to the result presented in [1], is expected. QC 20111201 NanoGate

We designed, manufactured, and integrated the first 3D microfluidic collector for capturing airborne particles directly into liquid; using an integrated electrostatic precipitator (ESP) sampler, designed for breath diagnostic purposes. The novel collector increases the sampler’s air-to-liquid capture efficiency by 35 times, when compared with previous 2D collectors. QC 20160722