Alternative nano-lithographic tools for shell-isolated nanoparticle enhanced Raman spectroscopy substrates.

Chemically synthesized metal nanoparticles (MNPs) have been widely used as surface-enhanced Raman spectroscopy (SERS) substrates for monitoring catalytic reactions. In some applications, however, the SERS MNPs, besides being plasmonically active, can also be catalytically active and result in Raman signals from undesired side products. The MNPs are typically insulated with a thin (∼3 nm), in principle pin-hole-free shell to prevent this. This approach, which is known as shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS), offers many advantages, such as better thermal and chemical stability of the plasmonic nanoparticle. However, having both a high enhancement factor and ensuring that the shell is pin-hole-free is challenging because there is a trade-off between the two when considering the shell thickness. So far in the literature, shell insulation has been successfully applied only to chemically synthesized MNPs. In this work, we alternatively study different combinations of chemical synthesis (bottom-up) and lithographic (top-down) routes to obtain shell-isolated plasmonic nanostructures that offer chemical sensing capabilities. The three approaches we study in this work include (1) chemically synthesized MNPs + chemical shell, (2) lithographic substrate + chemical shell, and (3) lithographic substrate + atomic layer deposition (ALD) shell. We find that ALD allows us to fabricate controllable and reproducible pin-hole-free shells. We showcase the ability to fabricate lithographic SHINER substrates which report an enhancement factor of 7.5 × 103 ± 17% for our gold nanodot substrates coated with a 2.8 nm aluminium oxide shell. Lastly, by introducing a gold etchant solution to our fabricated SHINER substrate, we verified that the shells fabricated with ALD are truly pin-hole-free.

Tracking the dynamics of thrombus formation in a blood vessel-on-chip with visible-light optical coherence tomography.

Thrombus formation is a physiological response to damage in a blood vessel that relies on a complex interplay of platelets, coagulation factors, immune cells, and the vessel wall. The dynamics of thrombus formation are essential for a deeper understanding of many disease processes, like bleeding, wound healing, and thrombosis. However, monitoring thrombus formation is challenging due to the limited imaging options available to analyze flowing blood. In this work, we use a visible-light optical coherence tomography (vis-OCT) system to monitor the dynamic process of the formation of thrombi in a microfluidic blood vessel-on-chip (VoC) device. Inside the VoC, thrombi form in a channel lined with a monolayer of endothelial cells and perfused by human whole blood. We show that the correlation of the vis-OCT signal can be utilized as a marker for thrombus formation. By thresholding the correlation during thrombus formation, we track and quantify the growth of the thrombi over time. We validate our results with fluorescence microscopic imaging of fibrin and platelet markers at the end of the blood perfusion assay. In conclusion, we demonstrate that the correlation of the vis-OCT signal can be used to visualize both the spatial and temporal behavior of the thrombus formation in flowing human whole blood.

Cell Patterning Technology on Polymethyl Methacrylate through Controlled Physicochemical and Biochemical Functionalization.

In recent years, innovative cell-based biosensing systems have been developed, showing impact in healthcare and life science research. Now, there is a need to design mass-production processes to enable their commercialization and reach society. However, current protocols for their fabrication employ materials that are not optimal for industrial production, and their preparation requires several chemical coating steps, resulting in cumbersome protocols. We have developed a simplified two-step method for generating controlled cell patterns on PMMA, a durable and transparent material frequently employed in the mass manufacturing of microfluidic devices. It involves air plasma and microcontact printing. This approach allows the formation of well-defined cell arrays on PMMA without the need for blocking agents to define the patterns. Patterns of various adherent cell types in dozens of individual cell cultures, allowing the regulation of cell-material and cell-cell interactions, were developed. These cell patterns were integrated into a microfluidic device, and their viability for more than 20 h under controlled flow conditions was demonstrated. This work demonstrated the potential to adapt polymeric cytophobic materials to simple fabrication protocols of cell-based microsystems, leveraging the possibilities for commercialization.

Controllable ingestion and release of guest components driven by interfacial molecular orientation of host liquid crystal droplets.

Controllable construction and manipulation of artificial multi-compartmental structures are crucial in understanding and imitating smart molecular elements such as biological cells and on-demand delivery systems. Here, we report a liquid crystal droplet (LCD) based three-dimensional system for controllable and reversible ingestion and release of guest aqueous droplets (GADs). Induced by interfacial thermodynamic fluctuation and internal topological defect, microscale LCDs with perpendicular anchoring condition at the interface would spontaneously ingest external components from the surroundings and transform them as radially assembled tiny GADs inside LCDs. Landau-de Gennes free-energy model is applied to describe and explain the assembly dynamics and morphologies of these tiny GADs, which presents a good agreement with experimental observations. Furthermore, the release of these ingested GADs can be actively triggered by changing the anchoring conditions at the interface of LCDs. Since those ingestion and release processes are controllable and happen very gently at room temperature and neutral pH environment without extra energy input, these microscale LCDs are very prospective to provide a unique and viable route for constructing hierarchical 3D structures with tunable components and compartments.

Low-Variance Surface-Enhanced Raman Spectroscopy Using Confined Gold Nanoparticles over Silicon Nanocones.

Surface-enhanced Raman spectroscopy (SERS) substrates are of utmost interest in the analyte detection of biological and chemical diagnostics. This is primarily due to the ability of SERS to sensitively measure analytes present in localized hot spots of the SERS nanostructures. In this work, we present the formation of 67 ± 6 nm diameter gold nanoparticles supported by vertically aligned shell-insulated silicon nanocones for ultralow variance SERS. The nanoparticles are obtained through discrete rotation glancing angle deposition of gold in an e-beam evaporating system. The morphology is assessed through focused ion beam tomography, energy-dispersive X-ray spectroscopy, and scanning electron microscopy. The optical properties are discussed and evaluated through reflectance measurements and finite-difference time-domain simulations. Lastly, the SERS activity is measured by benzenethiol functionalization and subsequent Raman spectroscopy in the surface scanning mode. We report a homogeneous analytical enhancement factor of 2.2 ± 0.1 × 107 (99% confidence interval for N = 400 grid spots) and made a comparison to other lithographically derived assemblies used in SERS. The strikingly low variance (4%) of our substrates facilitates its use for many potential SERS applications.

Automated assessment of human engineered heart tissues using deep learning and template matching for segmentation and tracking.

The high rate of drug withdrawal from the market due to cardiovascular toxicity or lack of efficacy, the economic burden, and extremely long time before a compound reaches the market, have increased the relevance of human in vitro models like human (patient-derived) pluripotent stem cell (hPSC)-derived engineered heart tissues (EHTs) for the evaluation of the efficacy and toxicity of compounds at the early phase in the drug development pipeline. Consequently, the EHT contractile properties are highly relevant parameters for the analysis of cardiotoxicity, disease phenotype, and longitudinal measurements of cardiac function over time. In this study, we developed and validated the software HAARTA (Highly Accurate, Automatic and Robust Tracking Algorithm), which automatically analyzes contractile properties of EHTs by segmenting and tracking brightfield videos, using deep learning and template matching with sub-pixel precision. We demonstrate the robustness, accuracy, and computational efficiency of the software by comparing it to the state-of-the-art method (MUSCLEMOTION), and by testing it with a data set of EHTs from three different hPSC lines. HAARTA will facilitate standardized analysis of contractile properties of EHTs, which will be beneficial for in vitro drug screening and longitudinal measurements of cardiac function.

Droplet microreactor for high-throughput fluorescence-based measurements of single catalyst particle acidity.

The particles of heterogeneous catalysts differ greatly in size, morphology, and most importantly, in activity. Studying these catalyst particles in batch typically results in ensemble averages, without any information at the level of individual catalyst particles. To date, the study of individual catalyst particles has been rewarding but is still rather slow and often cumbersome1. Furthermore, these valuable in-depth studies at the single particle level lack statistical relevance. Here, we report the development of a droplet microreactor for high-throughput fluorescence-based measurements of the acidities of individual particles in fluid catalytic cracking (FCC) equilibrium catalysts (ECAT). This method combines systematic screening of single catalyst particles with statistical relevance. An oligomerization reaction of 4-methoxystyrene, catalyzed by the Brønsted acid sites inside the zeolite domains of the ECAT particles, was performed on-chip at 95 °C. The fluorescence signal generated by the reaction products inside the ECAT particles was detected near the outlet of the microreactor. The high-throughput acidity screening platform was capable of detecting ~1000 catalyst particles at a rate of 1 catalyst particle every 2.4 s. The number of detected catalyst particles was representative of the overall catalyst particle population with a confidence level of 95%. The measured fluorescence intensities showed a clear acidity distribution among the catalyst particles, with the majority (96.1%) showing acidity levels belonging to old, deactivated catalyst particles and a minority (3.9%) exhibiting high acidity levels. The latter are potentially of high interest, as they reveal interesting new physicochemical properties indicating why the particles were still highly acidic and reactive.

Microfluidic Impedance Cytometry for Single-Cell Particulate Inorganic Carbon:Particulate Organic Carbon Measurements of Calcifying Algae.

Calcifying algae, like coccolithophores, greatly contribute to the oceanic carbon cycle and are therefore of particular interest for ocean carbon models. They play a key role in two processes that are important for the effective CO2 flux: The organic carbon pump (photosynthesis) and the inorganic carbon pump (calcification). The relative contribution of calcification and photosynthesis can be measured in algae by the amount of particulate inorganic carbon (PIC) and particulate organic carbon (POC). A microfluidic impedance cytometer is presented, enabling non-invasive and high-throughput assessment of the calcification state of single coccolithophore cells. Gradual modification of the exoskeleton by acidification results in a strong linear fit (R 2 = 0.98) between the average electrical phase and the PIC:POC ratio of the coccolithophore Emiliania huxleyi 920/9. The effect of different CO2 treatments on the PIC:POC ratio, however, is inconclusive, indicating that there is no strong effect observed for this particular strain. Lower PIC:POC ratios in cultures that grew to higher cell densities are found, which are also recorded with the impedance-based PIC:POC sensor. The development of this new quantification tool for small volumes paves the way for high-throughput analysis while applying multi-variable environmental stressors to support projections of the future marine carbon cycle.

Supervised machine learning in microfluidic impedance flow cytometry for improved particle size determination.

The assessment of particle and cell size in electrical microfluidic flow cytometers has become common practice. Nevertheless, in flow cytometers with coplanar electrodes accurate determination of particle size is difficult, owing to the inhomogeneous electric field. Pre-defined signal templates and compensation methods have been introduced to correct for this positional dependence, but are cumbersome when dealing with irregular signal shapes. We introduce a simple and accurate post-processing method without the use of pre-defined signal templates and compensation functions using supervised machine learning. We implemented a multiple linear regression model and show an average reduction of the particle diameter variation by 37% with respect to an earlier processing method based on a feature extraction algorithm and compensation function. Furthermore, we demonstrate its application in flow cytometry by determining the size distribution of a population of small (4.6 ± 0.9 µm) and large (5.9 ± 0.8 µm) yeast cells. The improved performance of this coplanar, two electrode chip enables precise cell size determination in easy to fabricate impedance flow cytometers.

Nanoparticle Printing for Microfluidic Applications: Bipolar Electrochemistry and Localized Raman Sensing Spots.

The local integration of metal nanoparticle films on 3D-structured polydimethylsiloxane (PDMS)-based microfluidic devices is of high importance for applications including electronics, electrochemistry, electrocatalysis, and localized Raman sensing. Conventional processes to locally deposit and pattern metal nanoparticles require multiple steps and shadow masks, or access to cleanroom facilities, and therefore, are relatively imprecise, or time and cost-ineffective. As an alternative, we present an aerosol-based direct-write method, in which patterns of nanoparticles generated via spark ablation are locally printed with sub-mm size and precision inside of microfluidic structures without the use of lithography or other masking methods. As proof of principle, films of Pt or Ag nanoparticles were printed in the chambers of a multiplexed microfluidic device and successfully used for two different applications: Screening electrochemical activity in a high-throughput fashion, and localized sensing of chemicals via surface-enhanced Raman spectroscopy (SERS). The versatility of the approach will enable the generation of functional microfluidic devices for applications that include sensing, high-throughput screening platforms, and microreactors using catalytically driven chemical conversions.

Multiplexed fluidic circuit board for controlled perfusion of 3D blood vessels-on-a-chip.

Three-dimensional (3D) blood vessels-on-a-chip (VoC) models integrate the biological complexity of vessel walls with dynamic microenvironmental cues, such as wall shear stress (WSS) and circumferential strain (CS). However, these parameters are difficult to control and are often poorly reproducible due to the high intrinsic diameter variation of individual 3D-VoCs. As a result, the throughput of current 3D systems is one-channel-at-a-time. Here, we developed a fluidic circuit board (FCB) for simultaneous perfusion of up to twelve 3D-VoCs using a single set of control parameters. By designing the internal hydraulic resistances in the FCB appropriately, it was possible to provide a pre-set WSS to all connected 3D-VoCs, despite significant variation in lumen diameters. Using this FCB, we found that variation of CS or WSS induce morphological changes to human induced pluripotent stem cell (hiPSC)-derived endothelial cells (ECs) and conclude that control of these parameters using a FCB is necessary to study 3D-VOCs.

The Effect of Dynamic, In Vivo-like Oxaliplatin on HCT116 Spheroids in a Cancer-on-Chip Model Is Representative of the Response in Xenografts.

The cancer xenograft model in which human cancer cells are implanted in a mouse is one of the most used preclinical models to test the efficacy of novel cancer drugs. However, the model is imperfect; animal models are ethically burdened, and the imperfect efficacy predictions contribute to high clinical attrition of novel drugs. If microfluidic cancer-on-chip models could recapitulate key elements of the xenograft model, then these models could substitute the xenograft model and subsequently surpass the xenograft model by reducing variation, increasing sensitivity and scale, and adding human factors. Here, we exposed HCT116 colorectal cancer spheroids to dynamic, in vivo-like, concentrations of oxaliplatin, including a 5 day drug-free period, on-chip. Growth inhibition on-chip was comparable to existing xenograft studies. Furthermore, immunohistochemistry showed a similar response in proliferation and apoptosis markers. While small volume changes in xenografts are hard to detect, in the chip-system, we could observe a temporary growth delay. Lastly, histopathology and a pharmacodynamic model showed that the cancer spheroid-on-chip was representative of the proliferating outer part of a HCT116 xenograft, thereby capturing the major driver of the drug response of the xenograft. Hence, the cancer-on-chip model recapitulated the response of HCT116 xenografts to oxaliplatin and provided additional drug efficacy information.

Contractility analysis of human engineered 3D heart tissues by an automatic tracking technique using a standalone application.

The use of Engineered Heart Tissues (EHT) as in vitro model for disease modeling and drug screening has increased, as they provide important insight into the genetic mechanisms, cardiac toxicity or drug responses. Consequently, this has highlighted the need for a standardized, unbiased, robust and automatic way to analyze hallmark physiological features of EHTs. In this study we described and validated a standalone application to analyze physiological features of EHTs in an automatic, robust, and unbiased way, using low computational time. The standalone application "EHT Analysis" contains two analysis modes (automatic and manual) to analyzes the contractile properties and the contraction kinetics of EHTs from high speed bright field videos. As output data, the graphs of displacement, contraction force and contraction kinetics per file will be generated together with the raw data. Additionally, it also generates a summary file containing all the data from the analyzed files, which facilitates and speeds up the post analysis. From our study we highlight the importance of analyzing the axial stress which is the force per surface area (µN/mm2). This allows to have a readout overtime of tissue compaction, axial stress and leave the option to calculate at the end point of an experiment the physiological cross-section area (PSCA). We demonstrated the utility of this tool by analyzing contractile properties and compaction over time of EHTs made out of a double reporter human pluripotent stem cell (hPSC) line (NKX2.5EGFP/+-COUP-TFIImCherry/+) and different ratios of human adult cardiac fibroblasts (HCF). Our standalone application "EHT Analysis" can be applied for different studies where the physiological features of EHTs needs to be analyzed under the effect of a drug compound or in a disease model.

A New Versatile Platform for Assessment of Improved Cardiac Performance in Human-Engineered Heart Tissues.

Cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) hold a great potential as human in vitro models for studying heart disease and for drug safety screening. Nevertheless, their associated immaturity relative to the adult myocardium limits their utility in cardiac research. In this study, we describe the development of a platform for generating three-dimensional engineered heart tissues (EHTs) from hPSC-CMs for the measurement of force while under mechanical and electrical stimulation. The modular and versatile EHT platform presented here allows for the formation of three tissues per well in a 12-well plate format, resulting in 36 tissues per plate. We compared the functional performance of EHTs and their histology in three different media and demonstrated that tissues cultured and maintained in maturation medium, containing triiodothyronine (T3), dexamethasone, and insulin-like growth factor-1 (TDI), resulted in a higher force of contraction, sarcomeric organization and alignment, and a higher and lower inotropic response to isoproterenol and nifedipine, respectively. Moreover, in this study, we highlight the importance of integrating a serum-free maturation medium in the EHT platform, making it a suitable tool for cardiovascular research, disease modeling, and preclinical drug testing.

Fluidic circuit board with modular sensor and valves enables stand-alone, tubeless microfluidic flow control in organs-on-chips.

Organs-on-chips are a unique class of microfluidic in vitro cell culture models, in which the in vivo tissue microenvironment is mimicked. Unfortunately, their widespread use is hampered by their operation complexity and incompatibility with end-user research settings. To address these issues, many commercial and non-commercial platforms have been developed for semi-automated culture of organs-on-chips. However, these organ-on-chip culture platforms each represent a closed ecosystem, with very little opportunity to interchange and integrate components from different platforms or to develop new ones. The translational organ-on-chip platform (TOP) is a multi-institutional effort to develop an open platform for automated organ-on-chip culture and integration of components from various developers. Central to TOP is the fluidic circuit board (FCB), a microfluidic plate with the form factor of a typical well plate. The FCB enables microfluidic control of multiple components like sensors or organ-on-chip devices through an interface based on openly available standards. Here, we report an FCB to integrate commercial and in-house developed components forming a stand-alone flow control system for organs-on-chips. The control system is able to achieve constant and pulsatile flow recirculation through a connected organ-on-chip device. We demonstrate that this system is able to automatically perfuse a heart-on-chip device containing co-cultures of cardiac tissues derived from human pluripotent stem cell-derived cardiomyocytes and monolayers of endothelial cells for five days. Altogether, we conclude that open technology platforms allow the integration of components from different sources to form functional and fit-for-purpose organ-on-chip systems. We anticipate that open platforms will play a central role in catalyzing and maturing further technological development of organ-on-chip culture systems.

Nanotechnology for a Sustainable Future: Addressing Global Challenges with the International Network4Sustainable Nanotechnology.

Nanotechnology has important roles to play in international efforts in sustainability. We discuss how current and future capabilities in nanotechnology align with and support the United Nations' Sustainable Development Goals. We argue that, as a field, we can accelerate the progress toward these goals both directly through technological solutions and through our special interdisciplinary skills in communication and tackling difficult challenges. We discuss the roles of targeting solutions, technology translation, the circular economy, and a number of examples from national efforts around the world in reaching these goals. We have formed a network of leading nanocenters to address these challenges globally and seek to recruit others to join us.

When You Do Not Get the Whole Picture: Scene Perception After Occipital Cortex Lesions.

Background: Occipital cortex lesions (OCLs) typically result in visual field defects (VFDs) contralateral to the damage. VFDs are usually mapped with perimetry involving the detection of point targets. This, however, ignores the important role of integration of visual information across locations in many tasks of everyday life. Here, we ask whether standard perimetry can fully characterize the consequences of OCLs. We compare performance on a rapid scene discrimination task of OCL participants and healthy observers with simulated VFDs. While the healthy observers will only suffer the loss of part of the visual scene, the damage in the OCL participants may further compromise global visual processing. Methods: VFDs were mapped with Humphrey perimetry, and participants performed two rapid scene discrimination tasks. In healthy participants, the VFDs were simulated with hemi- and quadrant occlusions. Additionally, the GIST model, a computational model of scene recognition, was used to make individual predictions based on the VFDs. Results: The GIST model was able to predict the performance of controls regarding the effects of the local occlusion. Using the individual predictions of the GIST model, we can determine that the variability between the OCL participants is much larger than the extent of the VFD could account for. The OCL participants can further be categorized as performing worse, the same, or better as their VFD would predict. Conclusions: While in healthy observers the extent of the simulated occlusion accounts for their performance loss, the OCL participants' performance is not fully determined by the extent or shape of their VFD as measured with Humphrey perimetry. While some OCL participants are indeed only limited by the local occlusion of the scene, for others, the lesions compromised the visual network in a more global and disruptive way. Yet one outperformed a healthy observer, suggesting a possible adaptation to the VFD. Preliminary analysis of neuroimaging data suggests that damage to the lateral geniculate nucleus and corpus callosum might be associated with the larger disruption of rapid scene discrimination. We believe our approach offers a useful behavioral tool for investigating why similar VFDs can produce widely differing limitations in everyday life.

Self-Propelled Detachment upon Coalescence of Surface Bubbles.

The removal of microbubbles from substrates is crucial for the efficiency of many catalytic and electrochemical gas evolution reactions in liquids. The current work investigates the coalescence and detachment of bubbles generated from catalytic decomposition of hydrogen peroxide. Self-propelled detachment, induced by the coalescence of two bubbles, is observed at sizes much smaller than those determined by buoyancy. Upon coalescence, the released surface energy is partly dissipated by the bubble oscillations, working against viscous drag. The remaining energy is converted to the kinetic energy of the out-of-plane jumping motion of the merged bubble. The critical ratio of the parent bubble sizes for the jumping to occur is theoretically derived from an energy balance argument and found to be in agreement with the experimental results. The present results provide both physical insight for the bubble interactions and practical strategies for applications in chemical engineering and renewable energy technologies like electrolysis.

Facilitating implementation of organs-on-chips by open platform technology.

Organ-on-chip (OoC) and multi-organs-on-chip (MOoC) systems have the potential to play an important role in drug discovery, disease modeling, and personalized medicine. However, most devices developed in academic labs remain at a proof-of-concept level and do not yet offer the ease-of-use, manufacturability, and throughput that are needed for widespread application. Commercially available OoC are easier to use but often lack the level of complexity of the latest devices in academia. Furthermore, researchers who want to combine different chips into MOoC systems are limited to one supplier, since commercial systems are not compatible with each other. Given these limitations, the implementation of standards in the design and operation of OoCs would strongly facilitate their acceptance by users. Importantly, the implementation of such standards must be carried out by many participants from both industry and academia to ensure a widespread acceptance and adoption. This means that standards must also leave room for proprietary technology development next to promoting interchangeability. An open platform with standardized interfacing and user-friendly operation can fulfill these requirements. In this Perspective article, the concept of an open platform for OoCs is defined from a technical perspective. Moreover, we discuss the importance of involving different stakeholders in the development, manufacturing, and application of such an open platform.

Determining Particle Size and Position in a Coplanar Electrode Setup Using Measured Opacity for Microfluidic Cytometry.

Microfluidic impedance flow cytometers enable high-throughput, non-invasive, and label-free detection of single-cells. Cytometers with coplanar electrodes are easy and cheap to fabricate, but are sensitive to positional differences of passing particles, owing to the inhomogeneous electric field. We present a novel particle height compensation method, which employs the dependence of measured electrical opacity on particle height. The measured electrical opacity correlates with the particle height as a result of the constant electrical double layer series capacitance of the electrodes. As an alternative to existing compensation methods, we use only two coplanar electrodes and multi-frequency analysis to determine the particle size of a mixture of 5, 6, and 7 µm polystyrene beads with an accuracy (CV) of 5.8%, 4.0%, and 2.9%, respectively. Additionally, we can predict the bead height with an accuracy of 1.5 µm (8% of channel height) using the measured opacity and we demonstrate its application in flow cytometry with yeast. The use of only two electrodes is of special interest for simplified, easy-to-use chips with a minimum amount of instrumentation and of limited size.