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.

Combined Numerical and Experimental Investigation of Localized Electroporation-Based Cell Transfection and Sampling.

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.

The desmoplakin-intermediate filament linkage regulates cell mechanics.

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.

Isolating single cells in a neurosphere assay using inertial microfluidics.

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.

Microfluidic device for stem cell differentiation and localized electroporation of postmitotic neurons.

New techniques to deliver 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.