A Three-Dimensional Printed Inertial Microfluidic Platform for Isolation of Minute Quantities of Vital Mitochondria.

We demonstrate a fast and easy-to-use three-dimensional printed microfluidic platform for mitochondria isolation from cell and tissue lysates based on inertial microfluidics. We present and quantify the quality of the isolated mitochondria by measuring the respiration rate under various conditions. We demonstrate that the technology produces vital mitochondria of equal quality to traditional, but more burdensome, differential centrifugation. We anticipate that the availability of improved tools for studies of bioenergetics to the broader biological community will enable these and other links to be explored in more meaningful ways, leading to further understanding of the links between energy, health, and disease.

Punishments and the dominance identity in networks.

In this study, we examine the effects of the structure of a network, the dominance identity of the actors, and the actors' ability to reward or punish on the use of punishment and the development of social bonds in a network. As expected, we find that peripheral rather than central actors in a network use punishment more, as do those with a high dominance identity. We also find that persons use punishment to increase or decrease their perceived level of dominance when their dominance identity is not verified. However, the patterns of interaction that develop in the network are an interactive function of network position, actor's dominance identity, and punishment use. We discuss how the social psychological drive to control one's identity interacts with social structural factors in the network to create different cultures of dyadic interaction.

The fairness identity and the emergence of inequality.

Social exchange theories explain how differences in structural power can generate inequalities in exchange networks. We argue here that even in the absence of structural power differences, inequality can emerge out of the identity process. We posit that when structurally equivalent actors are uncertain about the resource levels available for distribution, different levels of the fairness identity and responses to identity non-verification will influence how they negotiate for resources. Results from an experiment that varies the fairness identity level and the identity verification of actors in two different equal power exchange networks confirm this. Absent structural power differences, the level of the fairness identity, identity non-verification, and structure of the network mutually influence the distribution of resources such that some dyads earn as much as two and a half times more than others. We discuss our findings as they pertain to unearthing the processes by which group inequalities arise and persist.

The science identity and entering a science occupation.

The initiative to increase the number of students in STEM disciplines and train them for a science-related job is a current national focus. Using longitudinal panel data from a national study that followed underrepresented college students in STEM fields, we investigate the neglected role that social psychological processes play in influencing science activity among the young. We study the impact of identity processes related to being a science student on entering a science occupation. More broadly, we examine whether an identity formulated in one institutional setting (education) has effects that persist to another institutional setting (the economy). We find that the science identity positively impacts the likelihood of entering a science occupation. It also serves as a mediator for other factors that are related to educational success. This provides insight into how an identity can guide behavior to move persons into structural positions across institutional domains.

Microchambers with Solid-State Phosphorescent Sensor for Measuring Single Mitochondrial Respiration.

It is now well established that, even within a single cell, multiple copies of the mitochondrial genome may be present (genetic heteroplasmy). It would be interesting to develop techniques to determine if and to what extent this genetic variation results in functional variation from one mitochondrion to the next (functional heteroplasmy). Measuring mitochondrial respiration can reveal the organelles' functional capacity for Adenosine triphosphate (ATP) production and determine mitochondrial damage that may arise from genetic or age related defects. However, available technologies require significant quantities of mitochondria. Here, we develop a technology to assay the respiration of a single mitochondrion. Our "micro-respirometer" consists of micron sized chambers etched out of borofloat glass substrates and coated with an oxygen sensitive phosphorescent dye Pt(II) meso-tetra(pentafluorophenyl)porphine (PtTFPP) mixed with polystyrene. The chambers are sealed with a polydimethylsiloxane layer coated with oxygen impermeable Viton rubber to prevent diffusion of oxygen from the environment. As the mitochondria consume oxygen in the chamber, the phosphorescence signal increases, allowing direct determination of the respiration rate. Experiments with coupled vs. uncoupled mitochondria showed a substantial difference in respiration, confirming the validity of the microchambers as single mitochondrial respirometers. This demonstration could enable future high-throughput assays of mitochondrial respiration and benefit the study of mitochondrial functional heterogeneity, and its role in health and disease.

Super-Resolution Imaging of Voltages in the Interior of Individual, Vital Mitochondria.

We present super-resolution microscopy of isolated functional mitochondria, enabling real-time studies of structure and function (voltages) in response to pharmacological manipulation. Changes in mitochondrial membrane potential as a function of time and position can be imaged in different metabolic states (not possible in whole cells), created by the addition of substrates and inhibitors of the electron transport chain, enabled by the isolation of vital mitochondria. By careful analysis of structure dyes and voltage dyes (lipophilic cations), we demonstrate that most of the fluorescent signal seen from voltage dyes is due to membrane bound dyes, and develop a model for the membrane potential dependence of the fluorescence contrast for the case of super-resolution imaging, and how it relates to membrane potential. This permits direct analysis of mitochondrial structure and function (voltage) of isolated, individual mitochondria as well as submitochondrial structures in the functional, intact state, a major advance in super-resolution studies of living organelles.

Nanofluidic platform for single mitochondria analysis using fluorescence microscopy.

Using nanofluidic channels in PDMS of cross section 500 nm × 2 µm, we demonstrate the trapping and interrogation of individual, isolated mitochondria. Fluorescence labeling demonstrates the immobilization of mitochondria at discrete locations along the channel. Interrogation of mitochondrial membrane potential with different potential sensitive dyes (JC-1 and TMRM) indicates the trapped mitochondria are vital in the respiration buffer. Fluctuations of the membrane potential can be observed at the single mitochondrial level. A variety of chemical challenges can be delivered to each individual mitochondrion in the nanofluidic system. As sample demonstrations, increases in the membrane potential are seen upon introduction of OXPHOS substrates into the nanofluidic channel. Introduction of Ca(2+) into the nanochannels induces mitochondrial membrane permeabilization (MMP), leading to depolarization, observed at the single mitochondrial level. A variety of applications in cancer biology, stem cell biology, apoptosis studies, and high throughput functional metabolomics studies can be envisioned using this technology.

Three-dimensional transistor arrays for intra- and inter-cellular recording.

Electrical impulse generation and its conduction within cells or cellular networks are the cornerstone of electrophysiology. However, the advancement of the field is limited by sensing accuracy and the scalability of current recording technologies. Here we describe a scalable platform that enables accurate recording of transmembrane potentials in electrogenic cells. The platform employs a three-dimensional high-performance field-effect transistor array for minimally invasive cellular interfacing that produces faithful recordings, as validated by the gold standard patch clamp. Leveraging the high spatial and temporal resolutions of the field-effect transistors, we measured the intracellular signal conduction velocity of a cardiomyocyte to be 0.182 m s-1, which is about five times the intercellular velocity. We also demonstrate intracellular recordings in cardiac muscle tissue constructs and reveal the signal conduction paths. This platform could provide new capabilities in probing the electrical behaviours of single cells and cellular networks, which carries broad implications for understanding cellular physiology, pathology and cell-cell interactions.

Physical and Electrical Characterization of Synthesized Millimeter Size Single Crystal Graphene, Using Controlled Bubbling Transfer.

In this work, we have investigated the influence of the transfer process on the monocrystalline graphene in terms of quality, morphology and electrical properties by analyzing the data obtained from optical microscopy, scanning electron microscopy, Raman spectroscopy and electrical characterizations. The influence of Cu oxidation on graphene prior to the transfer is also discussed. Our results show that the controlled bubbling electrochemical delamination transfer is an easy and fast transfer technique suitable for transferring large single crystals graphene without degrading the graphene quality. Moreover, Raman spectroscopy investigation reveals that the Cu surface oxidation modifies the strain of the graphene film. We have observed that graphene laying on unoxidized Cu is subject to a biaxial strain in compression, while graphene on Cu oxide is subject to a biaxial strain in tension. However, after graphene was transferred to a host substrate, these strain effects were strongly reduced, leaving a homogeneous graphene on the substrate. The transferred single crystal graphene on silicon oxide substrate was used to fabricate transmission line method (TLM) devices. Electrical measurements show low contact resistance ~150 Ω·µm, which confirms the homogeneity and high quality of transferred graphene.

Demonstration and application of diffusive and ballistic wave propagation for drone-to-ground and drone-to-drone wireless communications.

In order to determine how an electromagnetic wave propagates from a base station to a cell phone or a wirelessly connected device, we use a novel Unmanned Aerial Vehicle (UAV) mapping technology to map the cellular network coverage at various altitudes in various terrains (flat, hilly, mountainous). For the flat terrains, the waves are shown to propagate ballistically: They have an altitude independent path loss consistent with minimal scatter in the propagation from transmitter to (aerial) receiver. In mountainous terrain, the waves are shown to propagate in the diffuse regime, and demonstrate a 10 dB increase in received signal intensity per 100' of altitude gain, up to 400'. In the intermediate case, evidence of coherent wave interference is clearly observed in altitude independent interference patterns. These general observations can be used to build a physical or empirical model for drone-to-ground and drone-to-drone propagation, for which existing models are shown to fail. While important for building physical models of wave propagation in wireless networks, this method can be used more generally to determine the magnitude and phase of an electromagnetic wave at every point in space, as well as usher in the era of drone-to-ground and drone-to-drone communications.

An ultra-high bandwidth nano-electronic interface to the interior of living cells with integrated fluorescence readout of metabolic activity.

We present the first ever broadband, calibrated electrical connection to the inside of a cell. The interior of a vital, living cell contains multiple dynamic and electrically active organelles such as mitochondria, chloroplasts, lysosomes, and the endoplasmic reticulum. However, little is known about the detailed electrical activity inside the cell. Here we show an ultra-high bandwidth nano-electronic interface to the interior of living cells with integrated fluorescence readout of metabolic activity. On-chip/on-petri dish nanoscale capacitance calibration standards are used to quantify the electronic coupling from bench to cell from DC to 26 GHz (with cell images at 22 GHz). The interaction of static to high frequency electromagnetic fields with the cell constituents induce currents of free charges and local reorganization of linked charges. As such, this enables a direct, calibrated, quantitative, nanoscale electronic interface to the interior of living cells. The interface could have a variety of applications in interfacing life sciences to nano-electronics, including electronic assays of membrane potential dynamics, nano-electronic actuation of cellular activity, and tomographic, nano-radar imaging of the morphology of vital organelles in the cytoplasm, during all phases of the cell life cycle (from development to senescence), under a variety of physiological environments, and under a broad suite of pharmacological manipulations.

Measurement of the combined quantum and electrochemical capacitance of a carbon nanotube.

The nature of the electronic interface between a nanotube and solvated ions in a liquid electrolyte is governed by two distinct physical phenomena: quantum and chemical. The quantum component arises from the sharply varying electronic density of states and the chemical component arises from ion screening and diffusion. Here, using an integrated on-chip shield technology, we measure the capacitance of one to a few nanotubes quantitatively as a function of both bias potential (from -0.7 V to 0.3 V) and ionic concentration (from 10 mM to 1 M KCl) at room temperature. We determine the relative contributions of the quantum and electrochemical capacitance, and confirm the measurements with theoretical models. This represents an important measurement of the quantum effects on capacitance in reduced dimensional systems in contact with liquid electrolytes, an important and emerging theme in the interface between nanotechnology, energy, and life.

Cardiac tissue engineering: state-of-the-art methods and outlook.

The purpose of this review is to assess the state-of-the-art fabrication methods, advances in genome editing, and the use of machine learning to shape the prospective growth in cardiac tissue engineering. Those interdisciplinary emerging innovations would move forward basic research in this field and their clinical applications. The long-entrenched challenges in this field could be addressed by novel 3-dimensional (3D) scaffold substrates for cardiomyocyte (CM) growth and maturation. Stem cell-based therapy through genome editing techniques can repair gene mutation, control better maturation of CMs or even reveal its molecular clock. Finally, machine learning and precision control for improvements of the construct fabrication process and optimization in tissue-specific clonal selections with an outlook of cardiac tissue engineering are also presented.

Sensing the electrical activity of single ion channels with top-down silicon nanoribbons.

Using top-down fabricated silicon nanoribbons, we measure the opening and closing of ion channels alamethicin and gramicidin A. A capacitive model of the system is proposed to demonstrate that the geometric capacitance of the nanoribbon is charged by ion channel currents. The integration of top-down nanoribbons with electrophysiology holds promise for integration of electrically active living systems with artificial electronics.

Scanning Microwave Microscopy of Vital Mitochondria in Respiration Buffer.

We demonstrate imaging using scanning microwave microscopy (SMM) of vital mitochondria in respiration buffer. The mitochondria are isolated from cultured HeLa cells and tethered to a solid graphene support. The mitochondria are kept vital (alive) using a respiration buffer, which provides nutrients to sustain the Krebs cycle. We verify that the mitochondria are "alive" by measuring the membrane potential using a voltage sensitive fluorescent dye (TMRE). The organelles are measured capacitively at 7 GHz. Several technical advances are demonstrated which enable this work: 1) The SMM operates in an electrophysiologically relevant liquid (hence conducting) environment; 2) The SMM operates in tapping mode, averaging the microwave reflection measurement over many tapping periods; 3) A tuned reflectometer enables increased sensitivity; 4) Variable frequencies up to 18 GHz are used; 5) In contrast with traditional matching/resonant methods that exhibit high quality factor that fail in the presence of liquids, interferometric/tuned reflectometer gives the possibility to adjust the quality factor or sensitivity even in the presence of the liquid.

Carbon-Nanotube-Electrolyte Interface: Quantum and Electric Double Layer Capacitance.

We present a comprehensive study of the electrochemical capacitance between a one-dimensional electronic material and an electrolyte. In contrast to a conventional, planar electrode, the nanoscale dimension of the electrode (with diameter smaller than the Debye length and approaching the size of the ions in solution) qualitatively changes the capacitance, which we measure and model herein. Furthermore, the finite density of states in these low dimensional electronic systems results in a quantum capacitance, which is comparable to the electrochemical capacitance. Using electrochemical impedance spectroscopy (EIS), we measure the ensemble average, complex, frequency dependent impedance (from 0.1 Hz to 1 MHz) between a purified (99.9%) semiconducting nanotube network and an aqueous electrolyte (KCl) at different concentrations between 10 mM and 1 M. The potential dependence of the capacitance is convoluted with the potential dependence of the in-plane conductance of the nanotube network, which we model using a transmission-line model to account for the frequency dependent in-plane impedance as well as the total interfacial impedance between the network and the electrolyte. The ionic strength dependence of the capacitance is expected to have a root cause from the double layer capacitance, which we model using a modified Poisson-Boltzmann equation. The relative contributions from those two capacitances can be quantitatively decoupled. We find a total capacitance per tube of 0.67-1.13 fF/µm according to liquid gate potential varying from -0.5 to -0.7 V.

Mitochondria, Bioenergetics and Apoptosis in Cancer.

Until recently, the dual roles of mitochondria in ATP production (bioenergetics) and apoptosis (cell life/death decision) were thought to be separate. New evidence points to a more intimate link between these two functions, mediated by the remodeling of the mitochondrial ultrastructure during apoptosis. While most of the key molecular players that regulate this process have been identified (primarily membrane proteins), the exact mechanisms by which they function are not yet understood. Because resistance to apoptosis is a hallmark of cancer, and because ultimately all chemotherapies are believed to result directly or indirectly in induction of apoptosis, a better understanding of the biophysical processes involved may lead to new avenues for therapy.

Versatile Bottom-Up Synthesis of Tethered Bilayer Lipid Membranes on Nanoelectronic Biosensor Devices.

Interfacing nanoelectronic devices with cell membranes can enable multiplexed detection of fundamental biological processes (such as signal transduction, electrophysiology, and import/export control) even down to the single ion channel level, which can lead to a variety of applications in pharmacology and clinical diagnosis. Therefore, it is necessary to understand and control the chemical and electrical interface between the device and the lipid bilayer membrane. Here, we develop a simple bottom-up approach to assemble tethered bilayer lipid membranes (tBLMs) on silicon wafers and glass slides, using a covalent tether attachment chemistry based on silane functionalization, followed by step-by-step stacking of two other functional molecular building blocks (oligo-poly(ethylene glycol) (PEG) and lipid). A standard vesicle fusion process was used to complete the bilayer formation. The monolayer synthetic scheme includes three well-established chemical reactions: self-assembly, epoxy-amine reaction, and EDC/NHS cross-linking reaction. All three reactions are facile and simple and can be easily implemented in many research labs, on the basis of common, commercially available precursors using mild reaction conditions. The oligo-PEG acts as the hydrophilic spacer, a key role in the formation of a homogeneous bilayer membrane. To explore the broad applicability of this approach, we have further demonstrated the formation of tBLMs on three common classes of (nano)electronic biosensor devices: indium-tin oxide-coated glass, silicon nanoribbon devices, and high-density single-walled carbon nanotubes (SWNT) networks on glass. More importantly, we incorporated alemethicin into tBLMs and realized the real-time recording of single ion channel activity with high sensitivity and high temporal resolution using the tBLMs/SWNT network transistor hybrid platform. This approach can provide a covalently bonded lipid coating on the oxide layer of nanoelectronic devices, which will enable a variety of applications in the emerging field of nanoelectronic interfaces to electrophysiology.

Resistive flow sensing of vital mitochondria with nanoelectrodes.

We report label-free detection of single mitochondria with high sensitivity using nanoelectrodes. Measurements of the conductance of carbon nanotube transistors show discrete changes of conductance as individual mitochondria flow over the nanoelectrodes in a microfluidic channel. Altering the bioenergetic state of the mitochondria by adding metabolites to the flow buffer induces changes in the mitochondrial membrane potential detected by the nanoelectrodes. During the time when mitochondria are transiently passing over the nanoelectrodes, this (nano) technology is sensitive to fluctuations of the mitochondrial membrane potential with a resolution of 10mV with temporal resolution of order milliseconds. Fluorescence based assays (in ideal, photon shot noise limited setups) are shown to be an order of magnitude less sensitive than this nano-electronic measurement technology. This opens a new window into the dynamics of an organelle critical to cellular function and fate.