Atomistic measurement and modeling of intrinsic fracture toughness of two-dimensional materials.

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 MoS2 and MoSe2 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).

Cellular Delivery of Large Functional Proteins and Protein-Nucleic Acid Constructs via Localized Electroporation.

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 (ß-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.

Scaling up single-cell mechanics to multicellular tissues - the role of the intermediate filament-desmosome network.

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 Ca2+-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.

Deep Learning-Assisted Automated Single Cell Electroporation Platform for Effective Genetic Manipulation of Hard-to-Transfect Cells.

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.

Edge-Mediated Annihilation of Vacancy Clusters in Monolayer Molybdenum Diselenide (MoSe2 ) under Electron Beam Irradiation.

Annihilation of vacancy clusters in monolayer molybdenum diselenide (MoSe2 ) 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 MoSe2 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.

High Throughput and Highly Controllable Methods for In Vitro Intracellular Delivery.

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.

Folding at the Microscale: Enabling Multifunctional 3D Origami-Architected Metamaterials.

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.

Temporal Sampling of Enzymes from Live Cells by Localized Electroporation and Quantification of Activity by SAMDI Mass Spectrometry.

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.

Nonlinear Mode Coupling and One-to-One Internal Resonances in a Monolayer WS2 Nanoresonator.

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 WS2 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.

Monoclonal Cell Line Generation and CRISPR/Cas9 Manipulation via Single-Cell Electroporation.

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.

Plasticity resulted from phase transformation for monolayer molybdenum disulfide film during nanoindentation simulations.

Molecular dynamics simulations on nanoindentation of circular monolayer molybdenum disulfide (MoS2) film are carried out to elucidate the deformation and failure mechanisms. Typical force-deflection curves are obtained, and in-plane stiffness of MoS2 is extracted according to a continuum mechanics model. The measured in-plane stiffness of monolayer MoS2 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 MoS2 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 MoS2, 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 MoS2 monolayer on the mechanical properties are also investigated.

Molecular-Level Engineering of Adhesion in Carbon Nanomaterial Interfaces.

Weak interfilament van der Waals interactions are potentially a significant roadblock in the development of carbon nanotube- (CNT-) and graphene-based nanocomposites. Chemical functionalization is envisioned as a means of introducing stronger intermolecular interactions at nanoscale interfaces, which in turn could enhance composite strength. This paper reports measurements of the adhesive energy of CNT-graphite interfaces functionalized with various coverages of arylpropionic acid. Peeling experiments conducted in situ in a scanning electron microscope show significantly larger adhesive energies compared to previously obtained measurements for unfunctionalized surfaces (Roenbeck et al. ACS Nano 2014, 8 (1), 124-138). Surprisingly, however, the adhesive energies are significantly higher when both surfaces have intermediate coverages than when one surface is densely functionalized. Atomistic simulations reveal a novel functional group interdiffusion mechanism, which arises for intermediate coverages in the presence of water. This interdiffusion is not observed when one surface is densely functionalized, resulting in energy trends that correlate with those observed in experiments. This unique intermolecular interaction mechanism, revealed through the integrated experimental-computational approach presented here, provides significant insights for use in the development of next-generation nanocomposites.

Single-cell detection of mRNA expression using nanofountain-probe electroporated molecular beacons.

New techniques for single-cell analysis enable new discoveries in gene expression and systems biology. Time-dependent measurements on individual cells are necessary, yet the common single-cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP-E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP-E is used to transfect a DNA-based beacon that detects glyceraldehyde 3-phosphate dehydrogenase and an RNA-based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time-dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.

Shear and friction between carbon nanotubes in bundles and yarns.

We perform a detailed density functional theory assessment of the factors that determine shear interactions between carbon nanotubes (CNTs) within bundles and in related CNT and graphene structures including yarns, providing an explanation for the shear force measured in recent experiments (Filleter, T. etal. Nano Lett. 2012, 12, 73). The potential energy barriers separating AB stacked structures are found to be irrelevant to the shear analysis for bundles and yarns due to turbostratic stacking, and as a result, the tube-tube shear strength for pristine CNTs is estimated to be <0.24 MPa, that is, extremely small. Instead, it is pinning due to the presence of defects and functional groups at the tube ends that primarily cause resistance to shear when bundles are fractured in weak vacuum (∼10(-5) Torr). Such defects and groups are estimated to involve 0.55 eV interaction energies on average, which is much larger than single-atom vacancy defects (approximately 0.039 eV). Furthermore, because graphitic materials are stiff they have large coherence lengths, and this means that push-pull effects result in force cancellation for vacancy and other defects that are internal to the CNTs. Another important factor is the softness of cantilever structures relative to the stiff CNTs in the experiments, as this contributes to elastic instability transitions that account for significant dissipation during shear that has been observed. The application of these results to the mechanical behavior of yarns is discussed, providing general guidelines for the manufacture of strong yarns composed of CNTs.

In situ electron microscopy four-point electromechanical characterization of freestanding metallic and semiconducting nanowires.

Electromechanical coupling is a topic of current interest in nanostructures, such as metallic and semiconducting nanowires, for a variety of electronic and energy applications. As a result, the determination of structure-property relations that dictate the electromechanical coupling requires the development of experimental tools to perform accurate metrology. Here, a novel micro-electro-mechanical system (MEMS) that allows integrated four-point, uniaxial, electromechanical measurements of freestanding nanostructures in-situ electron microscopy, is reported. Coupled mechanical and electrical measurements are carried out for penta-twinned silver nanowires, their resistance is identified as a function of strain, and it is shown that resistance variations are the result of nanowire dimensional changes. Furthermore, in situ SEM piezoresistive measurements on n-type, [111]-oriented silicon nanowires up to unprecedented levels of ∼7% strain are demonstrated. The piezoresistance coefficients are found to be similar to bulk values. For both metallic and semiconducting nanowires, variations of the contact resistance as strain is applied are observed. These variations must be considered in the interpretation of future two-point electromechanical measurements.

Nanofountain probe electroporation (NFP-E) of single cells.

The ability to precisely deliver molecules into single cells is of great interest to biotechnology researchers for advancing applications in therapeutics, diagnostics, and drug delivery toward the promise of personalized medicine. The use of bulk electroporation techniques for cell transfection has increased significantly in the past decade, but the technique is nonspecific and requires high voltage, resulting in variable efficiency and low cell viability. We have developed a new tool for electroporation using nanofountain probe (NFP) technology, which can deliver molecules into cells in a manner that is highly efficient and gentler to cells than bulk electroporation or microinjection. Here we demonstrate NFP electroporation (NFP-E) of single HeLa cells within a population by transfecting them with fluorescently labeled dextran and imaging the cells to evaluate the transfection efficiency and cell viability. Our theoretical analysis of the mechanism of NFP-E reveals that application of the voltage creates a localized electric field between the NFP cantilever tip and the region of the cell membrane in contact with the tip. Therefore, NFP-E can deliver molecules to a target cell with minimal effect of the electric potential on the cell. Our experiments on HeLa cells confirm that NFP-E offers single cell selectivity, high transfection efficiency (>95%), qualitative dosage control, and very high viability (92%) of transfected cells.

Individual GaN nanowires exhibit strong piezoelectricity in 3D.

Semiconductor GaN NWs are promising components in next generation nano- and optoelectronic systems. In addition to their direct band gap, they exhibit piezoelectricity, which renders them particularly attractive in energy harvesting applications for self-powered devices. Nanowires are often considered as one-dimensional nanostructures; however, the electromechanical coupling leads to a third rank tensor that for wurtzite crystals (GaN NWs) possesses three independent coefficients, d(33), d(13), and d(15). Therefore, the full piezoelectric characterization of individual GaN NWs requires application of electric fields in different directions and measurements of associated displacements on the order of several picometers. In this Letter, we present an experimental approach based on scanning probe microscopy to directly quantify the three-dimensional piezoelectric response of individual GaN NWs. Experimental results reveal that GaN NWs exhibit strong piezoelectricity in three dimensions, with up to six times the effect in bulk. Based on finite element modeling, this finding has major implication on the design of energy harvesting systems exhibiting unprecedented levels of power density production. The presented method is applicable to other piezoelectric NW materials as well as wires manufactured along different crystallographic orientations.

Experimental-computational study of shear interactions within double-walled carbon nanotube bundles.

The mechanical behavior of carbon nanotube (CNT)-based fibers and nanocomposites depends intimately on the shear interactions between adjacent tubes. We have applied an experimental-computational approach to investigate the shear interactions between adjacent CNTs within individual double-walled nanotube (DWNT) bundles. The force required to pull out an inner bundle of DWNTs from an outer shell of DWNTs was measured using in situ scanning electron microscopy methods. The normalized force per CNT-CNT interaction (1.7 ± 1.0 nN) was found to be considerably higher than molecular mechanics (MM)-based predictions for bare CNTs (0.3 nN). This MM result is similar to the force that results from exposure of newly formed CNT surfaces, indicating that the observed pullout force arises from factors beyond what arise from potential energy effects associated with bare CNTs. Through further theoretical considerations we show that the experimentally measured pullout force may include small contributions from carbonyl functional groups terminating the free ends of the CNTs, corrugation of the CNT-CNT interactions, and polygonization of the nanotubes due to their mutual interactions. In addition, surface functional groups, such as hydroxyl groups, that may exist between the nanotubes are found to play an unimportant role. All of these potential energy effects account for less than half of the ~1.7 nN force. However, partially pulled-out inner bundles are found not to pull back into the outer shell after the outer shell is broken, suggesting that dissipation is responsible for more than half of the pullout force. The sum of force contributions from potential energy and dissipation effects are found to agree with the experimental pullout force within the experimental error.