Microfluidic Device for Localized Electroporation

Electroporation is a common method of transfection due to its relatively low risk and high transfection efficiency. The most common method of electroporation is bulk electroporation which is easily performed on large quantities of cells yet results in variable levels of viability and transfection efficiency across the population. Localized electroporation is an alternative that can be administered on a similar scale but results in much more consistent with higher quality transfection and higher cell viability. This paper discusses the creation and use of a simple and cost-effective device using porous membrane for performing localized electroporation

Inactivation of SAG E3 Ubiquitin Ligase Blocks Embryonic Stem Cell Differentiation and Sensitizes Leukemia Cells to Retinoid Acid

Sensitive to Apoptosis Gene (SAG), also known as RBX2 (RING box protein-2), is the RING component of SCF (SKP1, Cullin, and F-box protein) E3 ubiquitin ligase. Our previous studies have demonstrated that SAG is an anti-apoptotic protein and an attractive anti-cancer target. We also found recently that Sag knockout sensitized mouse embryonic stem cells (mES) to radiation and blocked mES cells to undergo endothelial differentiation. Here, we reported that compared to wild-type mES cells, the Sag−/− mES cells were much more sensitive to all-trans retinoic acid (RA)-induced suppression of cell proliferation and survival. While wild-type mES cells underwent differentiation upon exposure to RA, Sag−/− mES cells were induced to death via apoptosis instead. The cell fate change, reflected by cellular stiffness, can be detected as early as 12 hrs post RA exposure by AFM (Atomic Force Microscopy). We then extended this novel finding to RA differentiation therapy of leukemia, in which the resistance often develops, by testing our hypothesis that SAG inhibition would sensitize leukemia to RA. Indeed, we found a direct correlation between SAG overexpression and RA resistance in multiple leukemia lines. By using MLN4924, a small molecule inhibitor of NEDD8-Activating Enzyme (NAE), that inactivates SAG-SCF E3 ligase by blocking cullin neddylation, we were able to sensitize two otherwise resistant leukemia cell lines, HL-60 and KG-1 to RA. Mechanistically, RA sensitization by MLN4924 was mediated via enhanced apoptosis, likely through accumulation of pro-apoptotic proteins NOXA and c-JUN, two well-known substrates of SAG-SCF E3 ligase. Taken together, our study provides the proof-of-concept evidence for effective treatment of leukemia patients by RA-MLN4924 combination

Optimization of Protein-Protein Interaction Measurements for Drug Discovery Using AFM Force Spectroscopy

Increasingly targeted in drug discovery, protein-protein interactions challenge current high throughput screening technologies in the pharmaceutical industry. Developing an effective and efficient method for screening small molecules or compounds is critical to accelerate the discovery of ligands for enzymes, receptors and other pharmaceutical targets. Here, we report developments of methods to increase the signal-to-noise ratio (SNR) for screening protein-protein interactions using atomic force microscopy (AFM) force spectroscopy. We have demonstrated the effectiveness of these developments on detecting the binding process between focal adhesion kinases (FAK) with protein kinase B (Akt1), which is a target for potential cancer drugs. These developments include optimized probe and substrate functionalization processes and redesigned probe-substrate contact regimes. Furthermore, a statistical-based data processing method was developed to enhance the contrast of the experimental data. Collectively, these results demonstrate the potential of the AFM force spectroscopy in automating drug screening with high throughput

Techniques to stimulate and interrogate cell–cell adhesion mechanics

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

Wearable Devices for Single-Cell Sensing and Transfection

Wearable healthcare devices are mainly used for biosensing and transdermal delivery. Recent advances in wearable biosensors allow for long-term and real-time monitoring of physiological conditions at a cellular resolution. Transdermal drug delivery systems have been further scaled down, enabling wide selections of cargo, from natural molecules (e.g., insulin and glucose) to bioengineered molecules (e.g., nanoparticles). Some emerging nanopatches show promise for precise single-cell gene transfection in vivo and have advantages over conventional tools in terms of delivery efficiency, safety, and controllability of delivered dose. In this review, we discuss recent technical advances in wearable micro/nano devices with unique capabilities or potential for single-cell biosensing and transfection in the skin or other organs, and suggest future directions for these fields. Highlights • Current wearable sensors have allowed for long-term, real-time detection of specific biomarkers directly from patients. • Miniaturized wearable biosensors with sensing elements interacting with skin or organs can capture target molecules from single cells, which results in significantly increased sensitivity, responding time, and precision. • Emerging wearable devices based on novel nanomaterials or nanofabrication show potential for single-cell detection in cancer cell screening, cardiomyocyte detection, and optogenetics. • Transdermal delivery devices have been scaled down to the micro- and/or nanoscale, and their applications have extended to wide selections of natural molecules and bioengineered molecules. • Emerging nanodevices show unique capabilities in precise single-cell gene transfection in vivo, with improved delivery efficiency, safety, and dose controllability

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

Airways hyperresponsiveness to different inhaled combination therapies in adolescent asthmatics

Local electrical characterization has wide spectrum of applications in various areas. However, there are a number of difficulties that hinder the precise measurement of local electrical properties of samples, particularly those within nano-scale spatial resolution. Inspired by these challenges, we developed a nano-robot enabled electrical characterization system that can be utilized to pinpoint the local electrical properties of materials, devices, and bioentities with high spatial and electrical resolution. This system consists of an electrical characterization unit and a nano-robot with an augment reality system, which was developed from a traditional atomic force microscopy (AFM). The augment reality system provides real-time visual feedback. The real-time visual display integrated with the real-time force feedback from the nano-robot allows a precise control of the position and force of the AFM tips towards samples, which are significant for the sensitivity of local electrical measurement. The system design and implementation are presented in the paper. Experiments were carried out to study the local conductance of a multi-wall carbon nanotube (MWCNT), demonstrating the effectiveness of this system. © 2012 IEEE.Link_to_subscribed_fulltex

On the Measurement of Energy Dissipation of Adhered Cells with the Quartz Microbalance with Dissipation Monitoring

We previously reported the finding of a linear correlation between the change of energy dissipation (ΔD) of adhered cells measured with the quartz crystal microbalance with dissipation monitoring (QCM-D) and the level of focal adhesions of the cells. To account for this correlation, we have developed a theoretical framework for assessing the ΔD-response of adhered cells. We rationalized that the mechanical energy of an oscillating QCM-D sensor coupled with a cell monolayer is dissipated through three main processes: the interfacial friction through the dynamic restructuring (formation and rupture) of cell-extracellular matrix (ECM) bonds, the interfacial viscous damping by the liquid trapped between the QCM-D sensor and the basal membrane of the cell layer, and the intracellular viscous damping through the viscous slip between the cytoplasm and stress fibers as well as among stress fibers themselves. Our modeling study shows that the interfacial viscous damping by the trapped liquid is the primary process for energy dissipation during the early stage of the cell adhesion, whereas the dynamic restructuring of cell-ECM bonds becomes more prevalent during the later stage of the cell adhesion. Our modeling study also establishes a positive linear correlation between the ΔD-response and the level of cell adhesion quantified with the number of cell-ECM bonds, which corroborates our previous experimental finding. This correlation with a wide well-defined linear dynamic range provides a much needed theoretical validation of the dissipation monitoring function of the QCM-D as a powerful quantitative analytical tool for cell study

Development of a Low Motion-Noise Humanoid Neck: Statics Analysis and Experimental Validation

Abstract-This paper presents our recently developed humanoid neck system that can effectively mimic motion of human neck with very low motion noises. The feature of low motion noises allows our system to work like a real human head/neck. Thus the level of acoustic noises from wearable equipments, such as donning respirators or chemical-resistant jackets, induced by human head motion can be simulated and investigated using such a system. The objective of this investigation is to facilitate using head-worn communication devices for the person who wears the protective equipment/uniform that usually produces communication-noise when the head/neck moves. Our low motion-noise humanoid neck system is based on the spring structure, which can generate 1 Degree of Freedom (DOF) jaw movement and 3DOF neck movement. To guarantee the low-noise feature, no noise-makers like gear and electrodriven parts are embedded in the head/neck structure. Instead, the motions are driven by seven cables, and the actuators pulling the cables are sealed in a sound insulation box. Furthermore, statics analysis of the system has been processed completely. Experimental results validate the analysis, and clearly show that the head/neck system can greatly mimic the motions of human head with an A-weighted noise level of 30 dB or below