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.

Desmoplakin connects desmosomal core components to intermediate filaments at sites of cell–cell adhesion. Modulating the strength of this linkage using desmoplakin mutants led to alterations in cell–substrate and cell–cell forces and cell stiffness as assessed by micropillar arrays and atomic force microscopy. Perturbation of the actin cytoskeleton leads to abrogation of these effects.

New techniques to deliver of nucleic acids and other molecules for gene editing and gene expression profiling, which can be performed with minimal perturbation to cell growth or differentiation, are essential for advancing biological research. Studying cells in their natural state, with temporal control, is particularly important for primary cells that are derived by differentiation from stem cells and are adherent, e.g., neurons. Existing high-throughput transfection methods either require cells to be in suspension or are highly toxic and limited to a single transfection per experiment. Here we present a microfluidic device that couples on-chip culture of adherent cells and transfection by localized electroporation. Integrated microchannels allow long-term cell culture on the device and repeated temporal transfection. The microfluidic device was validated by first performing electroporation of HeLa and HT1080 cells, with transfection efficiencies of ~95% for propidium iodide and up to 50% for plasmids. Application to primary cells was demonstrated by on-chip differentiation of neural stem cells and transfection of postmitotic neurons with a green fluorescent protein plasmid.

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.

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 MALDI (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 This article is protected by copyright. All rights reserved. 3 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 labelfree assay format, the LCAD with SAMDI enables a powerful new method for monitoring the dynamics of cellular activity from small populations of cells.

A biologically inspired, multilayer laminate structural design is deployed into nanocomposite fi lms of graphene oxide-poly(methyl methacrylate) (GO-PMMA). The resulting multilayer GO-PMMA fi lms show greatly enhanced mechanical properties compared to pure-graphene-oxide fi lms, with up to 100% increases in stiffness and strength when optimized. Notably, a new morphology is observed at fracture surfaces: whereas pure-graphene-oxide fi lms show clean fracture surfaces consistent with crack initiation and propagation perpendicular to the applied tensile load, the GO-PMMA multilayer laminates show terracing consistent with crack stopping and defl ection mechanisms. As a consequence, these macroscopic GO-PMMA fi lms become defect-tolerant and can maintain their tensile strengths as their sample volumes increase. Linear elastic fracture analysis supports these observations by showing that the stiffness modulation introduced by including PMMA layers within a graphene oxide fi lm can act to shield or defl ect cracks, thereby delaying failure and allowing the material to access more of its inherent strength. Together, these data clearly demonstrate that desirable defect-tolerant traits of structural biomaterials can indeed be incorporated into graphene-oxide-based nanocomposites.

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.

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.