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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 (FACS) 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 micro-confinement 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.

Annihilation of vacancy clusters in monolayer molybdenum diselenide (MoSe

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.

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.

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.

The International Technology Roadmap for Semiconductors (ITRS [ 1 ] ) identifi es emerging technologies with the potential to sustain Moore’s Law. A necessary succession from planar CMOS (complementary metal-oxide semiconductors) to nonplanar/dual-gate CMOS, and ultimately to novel architectures such as carbon nanotube (CNT)-based nano-electromechanical systems (NEMS) is envisioned. The ITRS also identifi es critical roadblocks currently precluding advances beyond CMOS. Primary among the roadblocks to NEMS are poor reliability and manufacturing challenges. Here we investigate the prevalent failure modes of CNT-based NEMS that hamper reliability through a combined experimental–computational approach. We fi rst identify their point of onset within the design space through in situ electromechanical characterization, highlighting the extremely limited region in which failure is avoided. We use dynamic multiphysics models to elucidate the underlying causes of failure, then return to the experimental characterization to show that the usable design space expands dramatically when employing novel electrode materials such as diamondlike carbon. Finally, we demonstrate the effi cacy of this solution through 100 successive actuation cycles without failure and applications to volatile memory operations. The immense potential of CNT-based NEMS is emergent in theoretical and experimental demonstrations of up to 100-GHz switching, [ 2 ] low leakage, and high ON–OFF ratios, [ 3 ] and outstanding current-carrying capacity. [ 4 , 5 ] To date however, individual demonstrations of performance such as these have been a primary focus, with limited reports of repeated actuation beyond a few cycles. [ 2 , 3 , 6 , 7 ] This is due

Nanodiamonds are rapidly emerging as promising carriers for next- generation therapeutics and drug delivery. However, developing future nanoscale devices and arrays that harness these nanoparticles will require unrealized spatial control. Furthermore, single-cell in vitro transfection methods lack an instrument that simultaneously offers the advantages of having nanoscale dimensions and control and continuous delivery via microfluidic components. To address this, two modes of controlled delivery of functionalized diamond nanoparticles are demonstrated using a broadly applicable nanofountain probe, a tool for direct-write nanopatterning with sub-100-nm resolution and direct in vitro single-cell injection. This study demonstrates the versatility of the nanofountain probe as a tool for high- fidelity delivery of functionalized nanodiamonds and other agents in nanomanufacturing and single-cell biological studies. These initial demonstrations of controlled delivery open the door to future studies examining the nanofountain probe's potential in delivering specific doses of DNA, viruses, and other therapeutically relevant biomolecules.

A previously proposed two-terminal carbon-nanotube-based device with closed-loop feedback is demonstrated through in situ scanning electron microscopy (SEM) experiments. The pull-in/pull-out tests were carried out using a multi-walled carbon nanotube (MWCNT) welded to a conductive probe attached to a nanomanipulator. The MWCNTs were cantilevered over a gold electrode and electrostatically actuated. The measured current-voltage curves exhibited the theoretically predicted hysteretic loop between the pull-in and pull-out processes. Both experiments and theoretical modeling demonstrated the bistability of the device confirming its utility in applications such as memory elements, NEMS switches, and logic devices. Failure mechanisms observed during the pull-in/pull-out event are also reported and discussed.

A monolithically integrated 3-D membrane or diaphragm/tip (called 3-D tip) of substantially all UNCD having a tip with a radius of about less than 50 nm capable of measuring forces in all three dimensions or being used as single tips or in large arrays for imprint of data on memory media, fabrication of nanodots of different materials on different substrates and many other uses such as nanolithography production of nanodots of biomaterials on substrates, etc. A method of molding UNCD is disclosed including providing a substrate with a predetermined pattern and depositing an oxide layer prior to depositing a carbide-forming metallic seed layer, followed by seeding with diamond nano or micropowder in solvent suspension, or mechanically polishing with diamond powder, or any other seeding method, followed by UNCD film growth conforming to the predetermined pattern. Thereafter, one or more steps of masking and/or etching and/or coating and/or selective removal and/or patterning and/or electroforming and/or lapping and/or polishing are used in any combination to form the tip or probe.