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Efficient and nontoxic delivery of foreign cargo into cells is a critical step in many biological studies and cell engineering workflows with applications in areas such as biomanufacturing and cell-based therapeutics. However, effective molecular delivery into cells involves optimizing several experimental parameters. In the case of electroporation-based intracellular delivery, there is a need to optimize parameters like pulse voltage, duration, buffer type, and cargo concentration for each unique application. Here, we present the protocol for fabricating and utilizing a high-throughput multi-well localized electroporation device (LEPD) assisted by deep learning-based image analysis to enable rapid optimization of experimental parameters for efficient and nontoxic molecular delivery into cells. The LEPD and the optimization workflow presented herein are relevant to both adherent and suspended cell types and different molecular cargo (DNA, RNA, and proteins). The workflow enables multiplexed combinatorial experiments and can be adapted to cell engineering applications requiring in vitro delivery. Key features • A high-throughput multi-well localized electroporation device (LEPD) that can be optimized for both adherent and suspended cell types. • Allows for multiplexed experiments combined with tailored pulse voltage, duration, buffer type, and cargo concentration. • Compatible with various molecular cargoes, including DNA, RNA, and proteins, enhancing its versatility for cell engineering applications. • Integration with deep learning-based image analysis enables rapid optimization of experimental parameters., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY license.)

The mechanical and thermal properties of transition metal dichalcogenides (TMDs) are directly relevant to their applications in electronics, thermoelectric devices, and heat management systems. In this study, we use a machine learning (ML) approach to parametrize molecular dynamics (MD) force fields to predict the mechanical and thermal transport properties of a library of monolayered TMDs (MoS 2 , MoTe 2 , WSe 2 , WS 2 , and ReS 2 ). The ML-trained force fields were then employed in equilibrium MD simulations to calculate the lattice thermal conductivities of the foregoing TMDs and to investigate how they are affected by small and large mechanical strains. Furthermore, using nonequilibrium MD, we studied thermal transport across grain boundaries. The presented approach provides a fast albeit accurate methodology to compute both mechanical and thermal properties of TMDs, especially for relatively large systems and spatially complex structures, where density functional theory computational cost is prohibitive.

Lattice-based constructs, often made by additive manufacturing, are attractive for many applications. Typically, such constructs are made from microscale or larger elements; however, smaller nanoscale components can lead to more unusual properties, including greater strength, lighter weight, and unprecedented resiliencies. Here, solid and hollow nanoparticles (nanoframes and nanocages; frame size: ~15 nanometers) were assembled into colloidal crystals using DNA, and their mechanical strengths were studied. Nanosolid, nanocage, and nanoframe lattices with identical crystal symmetries exhibit markedly different specific stiffnesses and strengths. Unexpectedly, the nanoframe lattice is approximately six times stronger than the nanosolid lattice. Nanomechanical experiments, electron microscopy, and finite element analysis show that this property results from the buckling, densification, and size-dependent strain hardening of nanoframe lattices. Last, these unusual open architectures show that lattices with structural elements as small as 15 nanometers can retain a high degree of strength, and as such, they represent target components for making and exploring a variety of miniaturized devices.

Delivery of proteins and protein-nucleic acid constructs into live cells enables a wide range of applications from gene editing to cell-based therapies and intracellular sensing. However, electroporation-based protein delivery remains challenging due to the large sizes of proteins, their low surface charge, and susceptibility to conformational changes that result in loss of function. Here, we use a nanochannel-based localized electroporation platform with multiplexing capabilities to optimize the intracellular delivery of large proteins (β-galactosidase, 472 kDa, 75.38% efficiency), protein-nucleic acid conjugates (protein spherical nucleic acids (ProSNA), 668 kDa, 80.25% efficiency), and Cas9-ribonucleoprotein complex (160 kDa, ∼60% knock-out and ∼24% knock-in) while retaining functionality post-delivery. Importantly, we delivered the largest protein to date using a localized electroporation platform and showed a nearly 2-fold improvement in gene editing efficiencies compared to previous reports. Furthermore, using confocal microscopy, we observed enhanced cytosolic delivery of ProSNAs, which may expand opportunities for detection and therapy.

Membrane disruption using Bulk Electroporation (BEP) is a widely used non-viral method for delivering biomolecules into cells. Recently, its microfluidic counterpart, Localized Electroporation (LEP), has been successfully used for several applications ranging from reprogramming and engineering cells for therapeutic purposes to non-destructive sampling from live cells for temporal analysis. However, the side effects of these processes on gene expression, that can affect the physiology of sensitive stem cells are not well understood. Here, we use single cell RNA sequencing (scRNA-seq) to investigate the effects of BEP and LEP on murine neural stem cell (NSC) gene expression. Our results indicate that unlike BEP, LEP does not lead to extensive cell death or activation of cell stress response pathways that may affect their long-term physiology. Additionally, our demonstrations show that LEP is suitable for multi-day delivery protocols as it enables better preservation of cell viability and integrity as compared to BEP., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Quantifying the intrinsic mechanical properties of two-dimensional (2D) materials is essential to predict the long-term reliability of materials and systems in emerging applications ranging from energy to health to next-generation sensors and electronics. Currently, measurements of fracture toughness and identification of associated atomistic mechanisms remain challenging. Herein, we report an integrated experimental-computational framework in which in-situ high-resolution transmission electron microscopy (HRTEM) measurements of the intrinsic fracture energy of monolayer MoS 2 and MoSe 2 are in good agreement with atomistic model predictions based on an accurately parameterized interatomic potential. Changes in crystalline structures at the crack tip and crack edges, as observed in in-situ HRTEM crack extension tests, are properly predicted. Such a good agreement is the result of including large deformation pathways and phase transitions in the parameterization of the inter-atomic potential. The established framework emerges as a robust approach to determine the predictive capabilities of molecular dynamics models employed in the screening of 2D materials, in the spirit of the materials genome initiative. Moreover, it enables device-level predictions with superior accuracy (e.g., fatigue lifetime predictions of electro- and opto-electronic nanodevices).

The emerging field of cell therapy offers the potential to treat and even cure a diverse array of diseases for which existing interventions are inadequate. Recent advances in micro and nanotechnology have added a multitude of single cell analysis methods to our research repertoire. At the same time, techniques have been developed for the precise engineering and manipulation of cells. Together, these methods have aided the understanding of disease pathophysiology, helped formulate corrective interventions at the cellular level, and expanded the spectrum of available cell therapeutic options. This review discusses how micro and nanotechnology have catalyzed the development of cell sorting, cellular engineering, and single cell analysis technologies, which have become essential workflow components in developing cell-based therapeutics. The review focuses on the technologies adopted in research studies and explores the opportunities and challenges in combining the various elements of cell engineering and single cell analysis into the next generation of integrated and automated platforms that can accelerate preclinical studies and translational research.

Manipulation of cells for applications such as biomanufacturing and cell-based therapeutics involves introducing biomolecular cargoes into cells. However, successful delivery is a function of multiple experimental factors requiring several rounds of optimization. Here, we present a high-throughput multiwell-format localized electroporation device (LEPD) assisted by deep learning image analysis that enables quick optimization of experimental factors for efficient delivery. We showcase the versatility of the LEPD platform by successfully delivering biomolecules into different types of adherent and suspension cells. We also demonstrate multicargo delivery with tight dosage distribution and precise ratiometric control. Furthermore, we used the platform to achieve functional gene knockdown in human induced pluripotent stem cells and used the deep learning framework to analyze protein expression along with changes in cell morphology. Overall, we present a workflow that enables combinatorial experiments and rapid analysis for the optimization of intracellular delivery protocols required for genetic manipulation.

Nondestructive cell membrane permeabilization systems enable the intracellular delivery of exogenous biomolecules for cell engineering tasks as well as the temporal sampling of cytosolic contents from live cells for the analysis of dynamic processes. Here, we report a microwell array format live-cell analysis device (LCAD) that can perform localized-electroporation induced membrane permeabilization, for cellular delivery or sampling, and directly interfaces with surface-based biosensors for analyzing the extracted contents. We demonstrate the capabilities of the LCAD via an automated high-throughput workflow for multimodal analysis of live-cell dynamics, consisting of quantitative measurements of enzyme activity using self-assembled monolayers for MALDI mass spectrometry (SAMDI) and deep-learning enhanced imaging and analysis. By combining a fabrication protocol that enables robust assembly and operation of multilayer devices with embedded gold electrodes and an automated imaging workflow, we successfully deliver functional molecules (plasmid and siRNA) into live cells at multiple time-points and track their effect on gene expression and cell morphology temporally . Furthermore, we report sampling performance enhancements, achieving saturation levels of protein tyrosine phosphatase activity measured from as few as 60 cells, and demonstrate control over the amount of sampled contents by optimization of electroporation parameters using a lumped model. Lastly, we investigate the implications of cell morphology on electroporation-induced sampling of fluorescent molecules using a deep-learning enhanced image analysis workflow.

Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting for cell isolation that can be harsh to sensitive cell types such as human-induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work, a fully automated version of the nanofountain probe electroporation (NFP-E) system, a nanopipette-based single-cell electroporation method is presented that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell-nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP-E is combined with microconfinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation., (© 2022 The Authors. Small published by Wiley-VCH GmbH.)

Annihilation of vacancy clusters in monolayer molybdenum diselenide (MoSe 2 ) under electron beam irradiation is reported. In situ high-resolution transmission electron microscopy observation reveals that the annihilation is achieved by diffusion of vacancies to the free edge near the vacancy clusters. Monte Carlo simulations confirm that it is energetically favorable for the vacancies to locate at the free edge. By computing the minimum energy path for the annihilation of one vacancy cluster as a case study, it is further shown that electron beam irradiation and pre-stress in the suspended MoSe 2 monolayer are necessary for the vacancies to overcome the energy barriers for diffusion. The findings suggest a new mechanism of vacancy healing in 2D materials and broaden the capability of electron beam for defect engineering of 2D materials, a promising way of tuning their properties for engineering applications., (© 2021 Wiley-VCH GmbH.)

Nature's masterfully synthesized biological materials take on greater relevance when viewed through the perspective of evolutionary abundance. The fact that beetles (order Coleoptera) account for a quarter of all extant lifeforms on Earth, makes them prime exponents of evolutionary success. In fact, their forewings are acknowledged as key traits to their radiative-adaptive success, which makes the beetle elytra a model structure for next-generation bioinspired synthetic materials. In this work, the multiscale morphological and mechanical characteristics of a variety of beetle species from the Cetoniinae subfamily are investigated with the aim of unraveling the underlying principles behind Nature's adaptation of the elytral bauplan to differences in body weight spanning three orders of magnitude. Commensurate with the integral implications of size variation in organisms, a combined material, morphological, and mechanical characterization framework, across spatial scales, was pursued. The investigation revealed the simultaneous presence of size-invariant strategies (chemical compositions, layered-fibrous architectures, graded motifs) as well as size-dependent features (scaling of elytral layers and characteristic dimensions of building blocks), synergistically combined to achieve similar levels of biomechanical functionality (stiffness, energy absorption, strength, deformation and toughening mechanisms) in response to developmental and selection constraints. The integral approach here presented seeks to shed light on Nature's solution to the problem of size variation, which underpins the diversity of beetles and the living world., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Single-cell delivery platforms like microinjection and nanoprobe electroporation enable unparalleled control over cell manipulation tasks but are generally limited in throughput. Here, we present an automated single-cell electroporation system capable of automatically detecting cells with artificial intelligence (AI) software and delivering exogenous cargoes of different sizes with uniform dosage. We implemented a fully convolutional network (FCN) architecture to precisely locate the nuclei and cytosol of six cell types with various shapes and sizes, using phase contrast microscopy. Nuclear staining or reporter fluorescence was used along with phase contrast images of cells within the same field of view to facilitate the manual annotation process. Furthermore, we leveraged the near-human inference capabilities of the FCN network in detecting stained nuclei to automatically generate ground-truth labels of thousands of cells within seconds, and observed no statistically significant difference in performance compared to training with manual annotations. The average detection sensitivity and precision of the FCN network were 95±1.7% and 90±1.8%, respectively, outperforming a traditional image-processing algorithm (72±7.2% and 72±5.5%) used for comparison. To test the platform, we delivered fluorescent-labeled proteins into adhered cells and measured a delivery efficiency of 90%. As a demonstration, we used the automated single-cell electroporation platform to deliver Cas9-guide RNA (gRNA) complexes into an induced pluripotent stem cell (iPSC) line to knock out a green fluorescent protein-encoding gene in a population of ~200 cells. The results demonstrate that automated single-cell delivery is a useful cell manipulation tool for applications that demand throughput, control, and precision.

Kirigami structures provide a promising approach to transform flat films into 3D complex structures that are difficult to achieve by conventional fabrication approaches. By designing the cutting geometry, it is shown that distinct buckling-induced out-of-plane configurations can be obtained, separated by a sharp transition characterized by a critical geometric dimension of the structures. In situ electron microscopy experiments reveal the effect of the ratio between the in-plane cut size and film thickness on out-of-plane configurations. Moreover, geometrically nonlinear finite element analyses (FEA) accurately predict the out-of-plane modes measured experimentally, their transition as a function of cut geometry, and provide the stress-strain response of the kirigami structures. The combined computational-experimental approach and results reported here represent a step forward in the characterization of thin films experiencing buckling-induced out-of-plane shape transformations and provide a path to control 3D configurations of micro- and nanoscale buckling-induced kirigami structures. The out-of-plane configurations promise great utility in the creation of micro- and nanoscale systems that can harness such structural behavior, such as optical scanning micromirrors, novel actuators, and nanorobotics. This work is of particular significance as the kirigami dimensions approach the sub-micrometer scale which is challenging to achieve with conventional micro-electromechanical system technologies., (© 2020 Wiley-VCH GmbH.)

In vitro and ex vivo intracellular delivery methods hold the key for releasing the full potential of tissue engineering, drug development, and many other applications. In recent years, there has been significant progress in the design and implementation of intracellular delivery systems capable of delivery at the same scale as viral transfection and bulk electroporation but offering fewer adverse outcomes. This review strives to examine a variety of methods for in vitro and ex vivo intracellular delivery such as flow-through microfluidics, engineered substrates, and automated probe-based systems from the perspective of throughput and control. Special attention is paid to a particularly promising method of electroporation using micro/nanochannel based porous substrates, which expose small patches of cell membrane to permeabilizing electric field. Porous substrate electroporation parameters discussed include system design, cells and cargos used, transfection efficiency and cell viability, and the electric field and its effects on molecular transport. The review concludes with discussion of potential new innovations which can arise from specific aspects of porous substrate-based electroporation platforms and high throughput, high control methods in general., (© 2020 Wiley-VCH GmbH.)

Introducing exogenous molecules into cells with high efficiency and dosage control is a crucial step in basic research as well as clinical applications. Here, the capability of the nanofountain probe electroporation (NFP-E) system to deliver proteins and plasmids in a variety of continuous and primary cell types with appropriate dosage control is reported. It is shown that the NFP-E can achieve fine control over the relative expression of two cotransfected plasmids. Finally, the dynamics of electropore closure after the pulsing ends with the NFP-E is investigated. Localized electroporation has recently been utilized to demonstrate the converse process of delivery (sampling), in which a small volume of the cytosol is retrieved during electroporation without causing cell lysis. Single-cell temporal sampling confers the benefit of monitoring the same cell over time and can provide valuable insights into the mechanisms underlying processes such as stem cell differentiation and disease progression. NFP-E parameters that maximize the membrane resealing time, which is essential for increasing the sampled volume and in meeting the challenge of monitoring low copy number biomarkers, are identified. Its application in CRISPR/Cas9 gene editing, stem cell reprogramming, and single-cell sampling studies is envisioned., (© 2020 Wiley-VCH GmbH.)

Mechanical metamaterials inspired by the Japanese art of paper folding have gained considerable attention because of their potential to yield deployable and highly tunable assemblies. The inherent foldability of origami structures enlarges the material design space with remarkable properties such as auxeticity and high deformation recoverability and deployability, the latter being key in applications where spatial constraints are pivotal. This work integrates the results of the design, 3D direct laser writing fabrication, and in situ scanning electron microscopic mechanical characterization of microscale origami metamaterials, based on the multimodal assembly of Miura-Ori tubes. The origami-architected metamaterials, achieved by means of microfabrication, display remarkable mechanical properties: stiffness and Poisson's ratio tunable anisotropy, large degree of shape recoverability, multistability, and even reversible auxeticity whereby the metamaterial switches Poisson's ratio sign during deformation. The findings here reported underscore the scalable and multifunctional nature of origami designs, and pave the way toward harnessing the power of origami engineering at small scales., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Measuring changes in enzymatic activity over time from small numbers of cells remains a significant technical challenge. In this work, a method for sampling the cytoplasm of cells is introduced to extract enzymes and measure their activity at multiple time points. A microfluidic device, termed the live cell analysis device (LCAD), is designed, where cells are cultured in microwell arrays fabricated on polymer membranes containing nanochannels. Localized electroporation of the cells opens transient pores in the cell membrane at the interface with the nanochannels, enabling extraction of enzymes into nanoliter-volume chambers. In the extraction chambers, the enzymes modify immobilized substrates, and their activity is quantified by self-assembled monolayers for matrix-assisted laser desorption/ionization (SAMDI) mass spectrometry. By employing the LCAD-SAMDI platform, protein delivery into cells is demonstrated. Next, it is shown that enzymes can be extracted, and their activity measured without a loss in viability. Lastly, cells are sampled at multiple time points to study changes in phosphatase activity in response to oxidation by hydrogen peroxide. With this unique sampling device and label-free assay format, the LCAD with SAMDI enables a powerful new method for monitoring the dynamics of cellular activity from small populations of cells., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cells and tissues sense, respond to and translate mechanical forces into biochemical signals through mechanotransduction, which governs individual cell responses that drive gene expression, metabolic pathways and cell motility, and determines how cells work together in tissues. Mechanotransduction often depends on cytoskeletal networks and their attachment sites that physically couple cells to each other and to the extracellular matrix. One way that cells associate with each other is through Ca 2+ -dependent adhesion molecules called cadherins, which mediate cell-cell interactions through adherens junctions, thereby anchoring and organizing the cortical actin cytoskeleton. This actin-based network confers dynamic properties to cell sheets and developing organisms. However, these contractile networks do not work alone but in concert with other cytoarchitectural elements, including a diverse network of intermediate filaments. This Review takes a close look at the intermediate filament network and its associated intercellular junctions, desmosomes. We provide evidence that this system not only ensures tissue integrity, but also cooperates with other networks to create more complex tissues with emerging properties in sensing and responding to increasingly stressful environments. We will also draw attention to how defects in intermediate filament and desmosome networks result in both chronic and acquired diseases., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Background: "Cuida tu Ánimo" (CTA [Take Care of Your Mood]) is an internet-based program for prevention and early intervention of adolescent depression implemented in Chile and Colombia. In the pilot application of the program, participants interacted with the program through a website that provided psychoeducational information, chat, and telephone appointments as well as monitoring and feedback messages. To date, most similar programs were not developed taking design features into consideration. The persuasive systems design (PSD) model is a comprehensive framework developed to aid in the design and evaluation of systems capable of influencing users' attitudes or behaviors. The purpose of this study was to evaluate the persuasiveness of CTA pilot program using the PSD model. Methods: The methodology used was expert evaluation, where specialists evaluate the program against a list of design principles. Results: Although the PSD model was not used to design the program, system features proposed by PSD were present, mainly "Dialogue support" features. Persuasion context analysis was not carried out by the developers. No aspects of the program could be related to "Primary task support" features because the developers did not define a primary task. Discussion: Key aspects of the PSD model could be incorporated in the CTA program to enhance system persuasiveness and improve adherence.

In the field of genetic engineering, the modification of genes to produce stable cell lines has a variety of applications ranging from the development of novel therapeutics to patient specific treatments. To successfully generate a cell line, the gene of interest must be delivered into the cell and integrated into the genome. The efficiency of cell line generation systems therefore depends on the efficiency of delivery of genetically modifying molecules such as plasmids and CRISPR/CAS9 complexes. In this work, we describe a localized electroporation-based system to generate stable monoclonal cell lines. By employing the nanofountain probe electroporation (NFP-E) system, single cells in patterned cultures are selectively transfected with plasmids, grown, and harvested to obtain stably expressing cell lines. Methods for microcontact printing, cell culture, electroporation, and harvesting are detailed in this chapter.

Graphene oxide (GO) sheets have been used as a model system to study how the mechanical properties of two-dimensional building blocks scale to their bulk form, such as paper-like, lamellar-structured thin films. Here, we report that the modulus of multilayer GO films can be significantly enhanced if some of the sheets are drastically weakened by introducing in-plane porosity. Nanometer-sized pores are introduced in GO sheets by chemical etching. Membrane-deflection measurements at the single-layer level show that the sheets are drastically weakened as the in-plane porosity increases. However, the mechanical properties of the corresponding multilayer films are much less sensitive to porosity. Surprisingly, the co-assembly of pristine and etched GO sheets yields even stiffer films than those made from pristine sheets alone. This is attributed to the more compliant nature of the soft porous sheets, which act as a binder to improve interlayer packing and load transfer in the multilayer films.

This paper describes a systematic study on the nanoscale toughening of monolayer graphene oxide (GO) by an ultra-thin polymer adlayer, which impedes the propagation of cracks during intraplanar fracture. Using molecular dynamics simulations, the crack-bridging capabilities of a library of five hydrogen-bonding-capable polymers are explored against an epoxide-rich GO substrate. The best crack-bridging effect is found in polymers with functional groups that can both donate/accept hydrogen atoms and have better capability to form cooperative hydrogen bonds. Aligning the chains of poly(acrylic acid) orthogonally to the crack propagation direction significantly enhances the fracture toughness of monolayer GO (by 310%) in comparison to that for an adlayer with randomly arranged chains (180% enhancement). Notably, van der Waals interactions, which are seldom highlighted in the fabrication of strong GO-polymer interfaces, are found to also provide significant crack-bridging capabilities when the polymers possess large side groups. These results pave the way for a set of design criteria that can help in remediating the intrinsically brittle mechanical behavior of two-dimensional materials, a barrier that currently restricts their potential applications.

Nanomechanical resonators make exquisite force sensors due to their small footprint, low dissipation, and high frequencies. Because the lowest resolvable force is limited by ambient thermal noise, resonators are either operated at cryogenic temperatures or coupled to a high-finesse optical or microwave cavity to reach sub aN Hz -1/2 sensitivity. Here, we show that operating a monolayer WS 2 nanoresonator in the strongly nonlinear regime can lead to comparable force sensitivities at room temperature. Cavity interferometry was used to transduce the nonlinear response of the nanoresonator, which was characterized by multiple pairs of 1:1 internal resonance. Some of the modes exhibited exotic line shapes due to the appearance of Hopf bifurcations, where the bifurcation frequency varied linearly with the driving force and forms the basis of the advanced sensing modality. The modality is less sensitive to the measurement bandwidth, limited only by the intrinsic frequency fluctuations, and therefore, advantageous in the detection of weak incoherent forces.

Whale baleen is a keratin-based biological material; it provides life-long (40-100 years) filter-feeding for baleen whales in place of teeth. This study reveals new aspects of the contribution of the baleen's hierarchical structure to its fracture toughness and connects it to the unique performance requirements, which require anisotropy of fracture resistance. Baleen plates are subjected to competing external effects of hydration and varying loading rates and demonstrate a high fracture toughness in transverse loading, which is the most important direction in the filtering function; in the longitudinal direction, the toughness is much lower since delamination and controlled flexure are expected and desirable. The compressive strength is also established and results support the fracture toughness measurements: it is also highly anisotropic, and exhibits a ductile-to-brittle transition with increasing strain rate in the dry condition, which is absent in the hydrated condition, conferring impact resistance to the baleen. Using 3D-printing prototypes that replicate the three principal structural features of the baleen plate (hollow medulla, mineralized tubules, and sandwich-tubular structure) are created, and the role of its structure in determining its mechanical behavior is demonstrated. These findings suggest new bioinspired engineering materials., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Localized electroporation has evolved as an effective technology for the delivery of foreign molecules into cells while preserving their viability. Consequently, this technique has potential applications in sampling the contents of live cells and the temporal assessment of cellular states at the single-cell level. Although there have been numerous experimental reports on localized electroporation-based delivery, a lack of a mechanistic understanding of the process hinders its implementation in sampling. In this work, we develop a multiphysics model that predicts the transport of molecules into and out of the cell during localized electroporation. Based on the model predictions, we optimize experimental parameters such as buffer conditions, electric field strength, cell confluency, and density of nanochannels in the substrate for successful delivery and sampling via localized electroporation. We also identify that cell membrane tension plays a crucial role in enhancing both the amount and the uniformity of molecular transport, particularly for macromolecules. We qualitatively validate the model predictions on a localized electroporation platform by delivering large molecules (bovine serum albumin and mCherry-encoding plasmid) and by sampling an exogeneous protein (tdTomato) in an engineered cell line.

Graphene oxide (GO), whose highly tunable surface chemistry enables the formation of strong interfacial hydrogen-bond networks, has garnered increasing interest in the design of devices that operate in the presence of water. For instance, previous studies have suggested that controlling GO's surface chemistry leads to enhancements in interfacial shear strength, allowing engineers to manage deformation pathways and control failure mechanisms. However, these previous reports have not explored the role of ambient humidity and only offer extensive chemical modifications to GO's surface as the main pathway to control GO's interfacial properties. Herein, through atomic force microscopy experiments on GO-GO interfaces, the adhesion energy and interfacial shear strength of GO were measured as a function of ambient humidity. Experimental evidence shows that adhesion energy and interfacial shear strength can be improved by a factor of 2-3 when GO is exposed to moderate (∼30% water weight) water content. Furthermore, complementary molecular dynamics simulations uncovered the mechanisms by which these nanomaterial interfaces achieve their properties. They reveal that the strengthening mechanism arises from the formation of strongly interacting hydrogen-bond networks, driven by the chemistry of the GO basal plane and intercalated water molecules between two GO surfaces. In summary, the methodology and findings here reported provide pathways to simultaneously optimize GO's interfacial and in-plane mechanical properties, by tailoring the chemistry of GO and accounting for water content, in engineering applications such as sensors, filtration membranes, wearable electronics, and structural materials.

Cell-cell adhesions maintain the mechanical integrity of multicellular tissues and have recently been found to act as mechanotransducers, translating mechanical cues into biochemical signals. Mechanotransduction studies have primarily focused on focal adhesions, sites of cell-substrate attachment. These studies leverage technical advances in devices and systems interfacing with living cells through cell-extracellular matrix adhesions. As reports of aberrant signal transduction originating from mutations in cell-cell adhesion molecules are being increasingly associated with disease states, growing attention is being paid to this intercellular signaling hub. Along with this renewed focus, new requirements arise for the interrogation and stimulation of cell-cell adhesive junctions. This review covers established experimental techniques for stimulation and interrogation of cell-cell adhesion from cell pairs to monolayers.

Stably transfected cell lines are widely used in drug discovery and biological research to produce recombinant proteins. Generation of these cell lines requires the isolation of multiple clones, using time-consuming dilution methods, to evaluate the expression levels of the gene of interest. A new and efficient method is described for the generation of monoclonal cell lines, without the need for dilution cloning. In this new method, arrays of patterned cell colonies and single cell transfection are employed to deliver a plasmid coding for a reporter gene and conferring resistance to an antibiotic. Using a nanofountain probe electroporation system, probe positioning is achieved through a micromanipulator with sub-micron resolution and resistance-based feedback control. The array of patterned cell colonies allows for rapid selection of numerous stably transfected clonal cell lines located on the same culture well, conferring a significant advantage over slower and labor-intensive traditional methods. In addition to plasmid integration, this methodology can be seamlessly combined with CRISPR/Cas9 gene editing, paving the way for advanced cell engineering., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pangolin scales form a durable armor whose hierarchical structure offers an avenue towards high performance bio-inspired materials design. In this study, the fracture resistance of African pangolin scales is examined using single edge crack three-point bend fracture testing in order to understand toughening mechanisms arising from the structures of natural mammalian armors. In these mechanical tests, the influence of material orientation and hydration level are examined. The fracture experiments reveal an exceptional fracture resistance due to crack deflection induced by the internal spatial orientation of lamellae. An order of magnitude increase in the measured fracture resistance due to scale hydration, reaching up to ~ 25kJ/m 2 was measured. Post-mortem analysis of the fracture samples was performed using a combination of optical and electron microscopy, and X-ray computerized tomography. Interestingly, the crack profile morphologies are observed to follow paths outlined by the keratinous lamellae structure of the pangolin scale. Most notably, the inherent structure of pangolin scales offers a pathway for crack deflection and fracture toughening. The results of this study are expected to be useful as design principles for high performance biomimetic applications., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Bighorn sheep (Ovis canadensis) rams hurl themselves at each other at speeds of ∼9 m/s (20 mph) to fight for dominance and mating rights. This necessitates impact resistance and energy absorption mechanisms, which stem from material-structure components in horns. In this study, the material hierarchical structure as well as correlations between the structure and mechanical properties are investigated. The major microstructural elements of horns are found as tubules and cell lamellae, which are oriented with (∼30⁰) angle with respect to each other. The cell lamellae contain keratin cells, in the shape of pancakes, possessing an average thickness of ∼2 µm and diameter of ∼20-30 µm. The morphology of keratin cells reveals the presence of keratin fibers and intermediate filaments with diameter of ∼200 nm and ∼12 nm, respectively, parallel to the cell surface. Quasi-static and high strain rate impact experiments, in different loading directions and hydration states, revealed a strong strain rate dependency for both dried and hydrated conditions. A strong anisotropy behavior was observed under impact for the dried state. The results show that the radial direction is the most preferable impact orientation because of its superior energy absorption. Detailed failure mechanisms under the aforementioned conditions are examined by bar impact recovery experiments. Shear banding, buckling of cell lamellae, and delamination in longitudinal and transverse direction were identified as the cause for strain softening under high strain rate impact. While collapse of tubules occurs in both quasi-static and impact tests, in radial and transverse directions, the former leads to more energy absorption and impact resistance., Statement of Significance: Bighorn sheep (Ovis canadensis) horns show remarkable impact resistance and energy absorption when undergoing high speed impact during the intraspecific fights. The present work illustrates the hierarchical structure of bighorn sheep horn at different length scales and investigates the energy dissipation mechanisms under different strain rates, loading orientations and hydration states. These results demonstrate how horn dissipates large amounts of energy, thus provide a new path to fabricate energy absorbent and crashworthiness engineering materials., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The translation of mechanical forces into biochemical signals plays a central role in guiding normal physiological processes during tissue development and homeostasis. Interfering with this process contributes to cardiovascular disease, cancer progression, and inherited disorders. The actin-based cytoskeleton and its associated adherens junctions are well-established contributors to mechanosensing and transduction machinery; however, the role of the desmosome-intermediate filament (DSM-IF) network is poorly understood in this context. Because a force balance among different cytoskeletal systems is important to maintain normal tissue function, knowing the relative contributions of these structurally integrated systems to cell mechanics is critical. Here we modulated the interaction between DSMs and IFs using mutant forms of desmoplakin, the protein bridging these structures. Using micropillar arrays and atomic force microscopy, we demonstrate that strengthening the DSM-IF interaction increases cell-substrate and cell-cell forces and cell stiffness both in cell pairs and sheets of cells. In contrast, disrupting the interaction leads to a decrease in these forces. These alterations in cell mechanics are abrogated when the actin cytoskeleton is dismantled. These data suggest that the tissue-specific variability in DSM-IF network composition provides an opportunity to differentially regulate tissue mechanics by balancing and tuning forces among cytoskeletal systems., (© 2017 Broussard, Yang, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Time-dependent mechanical characterization of nanowires is critical to understand their long-term reliability in applications, such as flexible-electronics and touch screens. It is also of great importance to develop a theoretical framework for experimentation and analysis on the mechanics of nanowires under time-dependent loading conditions, such as stress-relaxation and fatigue. Here, we combine in situ scanning electron microscope (SEM)/transmission electron microscope (TEM) tests with atomistic and phase-field simulations to understand the deformation mechanisms of single crystal silver nanowires held under constant strain. We observe that the nanowires initially undergo stress-relaxation, where the stress reduces with time and saturates after some time period. The stress-relaxation process occurs due to the formation of few dislocations and stacking faults. Remarkably, after a few hours the nanowires rupture suddenly. The reason for this abrupt failure of the nanowire was identified as stress-assisted diffusion, using phase-field simulations. Under a large applied strain, diffusion leads to the amplification of nanowire surface perturbation at long wavelengths and the nanowire fails at the stress-concentrated thin cross-sectional regions. An analytical analysis on the competition between the elastic energy and the surface energy predicts a longer time to failure for thicker nanowires than thinner ones, consistent with our experimental observations. The measured time to failure of nanowires under cyclic loading conditions can also be explained in terms of this mechanism.

Molecular dynamics simulations on nanoindentation of circular monolayer molybdenum disulfide (MoS 2 ) film are carried out to elucidate the deformation and failure mechanisms. Typical force-deflection curves are obtained, and in-plane stiffness of MoS 2 is extracted according to a continuum mechanics model. The measured in-plane stiffness of monolayer MoS 2 is about 182 ± 14 N m -1 , corresponding to an effective Young's modulus of 280 ± 21 GPa. More interestingly, at a critical indentation depth, the loading force decreases sharply and then increases again. The loading-unloading-reloading processes at different initial unloading deflections are also conducted to explain the phenomenon. It is found that prior to the critical depth, the monolayer MoS 2 film can return to the original state after completely unloading, while there is hysteresis when unloading after the critical depth and residual deformation exists after indenter fully retracted, indicating plasticity. This residual deformation is found to be caused by the changed lattice structure of the MoS 2 , i.e. a phase transformation. The critical pressure to induce the phase transformation is then calculated to be 36 ± 2 GPa, consistent with other studies. Finally, the influences of temperature, the diameter and indentation rate of MoS 2 monolayer on the mechanical properties are also investigated.

Several recent micro- and nanotechnologies have provided novel methods for biological studies of adherent cells because the small features of these new biotools provide unique capabilities for accessing cells without the need for suspension or lysis. These novel approaches have enabled gentle but effective delivery of molecules into specific adhered target cells, with unprecedented spatial resolution. We review here recent progress in the development of these technologies with an emphasis on in vitro delivery into adherent cells utilizing mechanical penetration or electroporation. We discuss the major advantages and limitations of these approaches and propose possible strategies for improvements. Finally, we discuss the impact of these technologies on biological research concerning cell-specific temporal studies, for example non-destructive sampling and analysis of intracellular molecules., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The mechanical properties of graphene oxide (GO) are of great importance for applications in materials engineering. Previous mechanochemical studies of GO typically focused on the influence of the degree of oxidation on the mechanical behavior. In this study, using density functional-based tight binding simulations, validated using density functional theory simulations, we reveal that the deformation and failure of GO are strongly dependent on the relative concentrations of epoxide (-O-) and hydroxyl (-OH) functional groups. Hydroxyl groups cause GO to behave as a brittle material; by contrast, epoxide groups enhance material ductility through a mechanically driven epoxide-to-ether functional group transformation. Moreover, with increasing epoxide group concentration, the strain to failure and toughness of GO significantly increases without sacrificing material strength and stiffness. These findings demonstrate that GO should be treated as a versatile, tunable material that may be engineered by controlling chemical composition, rather than as a single, archetypical material.

Background: Major depression is a highly prevalent and severe mental disease. Despite the effective treatment options available, the risk of relapse is high. Interventions based on information and communication technologies generate innovative opportunities to provide support to patients after they completed treatment for depression., Materials and Methods: This acceptability study evaluated the Internet-based program Apoyo, Seguimiento y Cuidado de Enfermedades a partir de Sistemas Operativos (ASCENSO) in terms of its feasibility and acceptability in a sample of 35 patients in Chile., Results: The study reveals high rates of acceptance and satisfaction among patients who actively used the program. As obstacles, patients mentioned technical problems, a lack of contact with other participants, and an insufficient connection between the program and the health service professionals., Conclusions: ASCENSO appears to be a promising complement to regular care for depression. Following improvements of the program based on participants' feedback, future research should evaluate its efficacy and cost-effectiveness.

Understanding the deformation mechanisms in multilayer graphene (MLG), an attractive material used in nanodevices as well as in the reinforcement of nanocomposites, is critical yet challenging due to difficulties in experimental characterization and the spatiotemporal limitations of atomistic modeling. In this study, we combine nanomechanical experiments with coarse-grained molecular dynamics (CG-MD) simulations to elucidate the mechanisms of deformation and failure of MLG sheets. Elastic properties of graphene sheets with one to three layers are measured using film deflection tests. A nonlinear behavior in the force vs deflection curves for MLGs is observed in both experiments and simulations: during loading/unloading cycles, MLGs dissipate energy through a "recoverable slippage" mechanism. The CG-MD simulations further reveal an atomic level interlayer slippage process and suggest that the dissipated energy scales with film perimeter. Moreover, our study demonstrates that the finite shear strength between individual layers could explain the experimentally measured size-dependent strength with thickness scaling in MLG sheets.

Sphere forming assays are routinely used for in vitro propagation and differentiation of stem cells. Because the stem cell clusters can become heterogeneous and polyclonal, they must first be dissociated into a single cell suspension for further clonal analysis or differentiation studies. The dissociated population is marred by the presence of doublets, triplets and semi-cleaved/intact clusters which makes identification and further analysis of differentiation pathways difficult. In this work, we use inertial microfluidics to separate the single cells and clusters in a population of chemically dissociated neurospheres. In contrast to previous microfluidic sorting technologies which operated at high flow rates, we implement the spiral microfluidic channel in a novel focusing regime that occurs at lower flow rates. In this regime, the curvature-induced Dean's force focuses the smaller, single cells towards the inner wall and the larger clusters towards the center. We further demonstrate that sorting in this low flow rate (and hence low shear stress) regime yields a high percentage (>90%) of viable cells and preserves multipotency by differentiating the sorted neural stem cell population into neurons and astrocytes. The modularity of the device allows easy integration with other lab-on-a-chip devices for upstream mechanical dissociation and downstream high-throughput clonal analysis, localized electroporation and sampling. Although demonstrated in the case of the neurosphere assay, the method is equally applicable to other sphere forming assays.

MEMS and other lab-on-a-chip systems are emerging as attractive alternatives to carry out experiments in situ the electron microscope. However, several electrical connections are usually required for operating these setups. Such connectivity is challenging inside the limited space of the TEM side-entry holder. Here, we design, implement and demonstrate a double-tilt TEM holder with capabilities for up to 9 electrical connections, operating in a high-resolution TEM. We describe the operating principle of the tilting and connection mechanisms and the physical implementation of the holder. To demonstrate the holder capabilities, we calibrate the tilting action, which has limits of ±15°, and establish the insulation resistance of the electronics to be 36GΩ, appropriate for measurements of currents down to the nano-amp (nA) regime. Furthermore, we demonstrate tensile testing of silver nanowires using a previously developed MEMS device for mechanical testing, using the implemented holder as the platform for electronic operation and sensing. The implemented holder can potentially have broad application to other areas where MEMS or electrically-actuated setups are used to carry out in situ TEM experiments., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The ability to bias chemical reaction pathways is a fundamental goal for chemists and material scientists to produce innovative materials. Recently, two-dimensional materials have emerged as potential platforms for exploring novel mechanically activated chemical reactions. Here we report a mechanochemical phenomenon in graphene oxide membranes, covalent epoxide-to-ether functional group transformations that deviate from epoxide ring-opening reactions, discovered through nanomechanical experiments and density functional-based tight binding calculations. These mechanochemical transformations in a two-dimensional system are directionally dependent, and confer pronounced plasticity and damage tolerance to graphene oxide monolayers. Additional experiments on chemically modified graphene oxide membranes, with ring-opened epoxide groups, verify this unique deformation mechanism. These studies establish graphene oxide as a two-dimensional building block with highly tuneable mechanical properties for the design of high-performance nanocomposites, and stimulate the discovery of new bond-selective chemical transformations in two-dimensional materials.

Rough surfaces immersed under water remain practically dry if the liquid-solid contact is on roughness peaks, while the roughness valleys are filled with gas. Mechanisms that prevent water from invading the valleys are well studied. However, to remain practically dry under water, additional mechanisms need consideration. This is because trapped gas (e.g. air) in the roughness valleys can dissolve into the water pool, leading to invasion. Additionally, water vapor can also occupy the roughness valleys of immersed surfaces. If water vapor condenses, that too leads to invasion. These effects have not been investigated, and are critically important to maintain surfaces dry under water. In this work, we identify the critical roughness scale, below which it is possible to sustain the vapor phase of water and/or trapped gases in roughness valleys - thus keeping the immersed surface dry. Theoretical predictions are consistent with molecular dynamics simulations and experiments.