Transfer printing is a method to transfer solid micro/nanoscale materials (herein called ‘inks’) from a substrate where they are generated to a different substrate by utilizing elastomeric stamps. Transfer printing enables the integration of heterogeneous materials to fabricate unexampled structures or functional systems that are found in recent advanced devices such as flexible and stretchable solar cells and LED arrays. While transfer printing exhibits unique features in material assembly capability, the use of adhesive layers or the surface modification such as deposition of self-assembled monolayer (SAM) on substrates for enhancing printing processes hinders its wide adaptation in microassembly of microelectromechanical system (MEMS) structures and devices. To overcome this shortcoming, we developed an advanced mode of transfer printing which deterministically assembles individual microscale objects solely through controlling surface contact area without any surface alteration. The absence of an adhesive layer or other modification and the subsequent material bonding processes ensure not only mechanical bonding, but also thermal and electrical connection between assembled materials, which further opens various applications in adaptation in building unusual MEMS devices.
28 Related JoVE Articles!
Mouse Genome Engineering Using Designer Nucleases
Institutions: University of Zurich, University of Minnesota.
Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro
transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.
Genetics, Issue 86, Oocyte microinjection, Designer nucleases, ZFN, TALEN, Genome Engineering
Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction
Institutions: Rutgers University, Koç University, New York University, Fairfield University.
We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be controlled by one computer.
Behavior, Issue 84, genetics, cognitive mechanisms, behavioral screening, learning, memory, timing
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun
Institutions: Harvard University, Harvard Medical School, Delft University of Technology.
In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5’ mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11
, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana
epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.
Environmental Sciences, Issue 86, Plant Leaves, Synthetic Biology, Plants, Genetically Modified, DNA, Plant, RNA, Gene Targeting, Plant Physiological Processes, Genes, Gene gun, Gibson assembly, Nicotiana benthamiana, Alternative splicing, confocal microscopy, chloroplast, peroxisome
A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Institutions: Dartmouth College, University of Rhode Island, Dartmouth College.
Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2
. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5
. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.
Biochemistry, Issue 85, Immunoassay, Protein Immunogenicity, MHC II, T cell epitope, High Throughput Screen, Deimmunization, Vaccine Design
Confocal Imaging of Confined Quiescent and Flowing Colloid-polymer Mixtures
Institutions: University of Houston.
The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed assembly1-3
, drug delivery4
, improved hydrocarbon recovery5-7
, and flowable electrodes for energy storage8
. Suspensions containing fluorescent colloids and non-adsorbing polymers are appealing model systems, as the ratio of the polymer radius of gyration to the particle radius and concentration of polymer control the range and strength of the interparticle attraction, respectively. By tuning the polymer properties and the volume fraction of the colloids, colloid fluids, fluids of clusters, gels, crystals, and glasses can be obtained9
. Confocal microscopy, a variant of fluorescence microscopy, allows an optically transparent and fluorescent sample to be imaged with high spatial and temporal resolution in three dimensions. In this technique, a small pinhole or slit blocks the emitted fluorescent light from regions of the sample that are outside the focal volume of the microscope optical system. As a result, only a thin section of the sample in the focal plane is imaged. This technique is particularly well suited to probe the structure and dynamics in dense colloidal suspensions at the single-particle scale: the particles are large enough to be resolved using visible light and diffuse slowly enough to be captured at typical scan speeds of commercial confocal systems10
. Improvements in scan speeds and analysis algorithms have also enabled quantitative confocal imaging of flowing suspensions11-16,37
. In this paper, we demonstrate confocal microscopy experiments to probe the confined phase behavior and flow properties of colloid-polymer mixtures. We first prepare colloid-polymer mixtures that are density- and refractive-index matched. Next, we report a standard protocol for imaging quiescent dense colloid-polymer mixtures under varying confinement in thin wedge-shaped cells. Finally, we demonstrate a protocol for imaging colloid-polymer mixtures during microchannel flow.
Chemistry, Issue 87, confocal microscopy, particle tracking, colloids, suspensions, confinement, gelation, microfluidics, image correlation, dynamics, suspension flow
High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli
Institutions: Aix-Marseille Université, Commissariat à l'énergie atomique et aux énergies alternatives (CEA) Saclay, France.
Escherichia coli (E. coli)
is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli
cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.
Bioengineering, Issue 89, E. coli, expression, recombinant, high throughput (HTP), purification, auto-induction, immobilized metal affinity chromatography (IMAC), tobacco etch virus protease (TEV) cleavage, disulfide bond isomerase C (DsbC) fusion, disulfide bonds, animal venom proteins/peptides
From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data
Institutions: Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory.
Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g.
, signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.
The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.
Bioengineering, Issue 90, 3D electron microscopy, feature extraction, segmentation, image analysis, reconstruction, manual tracing, thresholding
Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches
Institutions: Centre National de la Recherche Scientifique (CNRS), Institut d'Ecologie et des Sciences de l'Environnement de Paris, Institut Pasteur.
Robots designed to track chemical leaks in hazardous industrial facilities1
or explosive traces in landmine fields2
face the same problem as insects foraging for food or searching for mates3
: the olfactory search is constrained by the physics of turbulent transport4
. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6
, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7
but also chemicals that are relevant to humans, e.g.
, volatile compounds emanating from cancer cells8
or toxic and illicit substances9-11
. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ
in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12
. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13
. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14
or using nanostructured gas sensors that mimic insect antennae15
Neuroscience, Issue 90, robotics, electroantennogram, EAG, gas sensor, electronic nose, olfactory search, surge and casting, moth, insect, olfaction, neuron
Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology
Institutions: DNA Medicine Institute, Harvard Medical School, NASA Glenn Research Center, ZIN Technologies.
Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.
Cellular Biology, Issue 93, Point-of-care, prototype, diagnostics, spaceflight, reduced gravity, parabolic flight, flow cytometry, fluorescence, cell counting, micromixing, spiral-vortex, blood mixing
Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding
Institutions: University of California, San Francisco, University of California, San Francisco, University of Southern California.
Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.
Bioengineering, Issue 88, liquid injection molding, reaction injection molding, molds, 3D printing, fused deposition modeling, rapid prototyping, medical devices, low cost, low volume, rapid turnaround time.
The Preparation of Electrohydrodynamic Bridges from Polar Dielectric Liquids
Institutions: Wetsus - Centre of Excellence for Sustainable Water Technology, IRCAM GmbH, Graz University of Technology.
Horizontal and vertical liquid bridges are simple and powerful tools for exploring the interaction of high intensity electric fields (8-20 kV/cm) and polar dielectric liquids. These bridges are unique from capillary bridges in that they exhibit extensibility beyond a few millimeters, have complex bi-directional mass transfer patterns, and emit non-Planck infrared radiation. A number of common solvents can form such bridges as well as low conductivity solutions and colloidal suspensions. The macroscopic behavior is governed by electrohydrodynamics and provides a means of studying fluid flow phenomena without the presence of rigid walls. Prior to the onset of a liquid bridge several important phenomena can be observed including advancing meniscus height (electrowetting), bulk fluid circulation (the Sumoto effect), and the ejection of charged droplets (electrospray). The interaction between surface, polarization, and displacement forces can be directly examined by varying applied voltage and bridge length. The electric field, assisted by gravity, stabilizes the liquid bridge against Rayleigh-Plateau instabilities. Construction of basic apparatus for both vertical and horizontal orientation along with operational examples, including thermographic images, for three liquids (e.g.
, water, DMSO, and glycerol) is presented.
Physics, Issue 91, floating water bridge, polar dielectric liquids, liquid bridge, electrohydrodynamics, thermography, dielectrophoresis, electrowetting, Sumoto effect, Armstrong effect
Picoinjection of Microfluidic Drops Without Metal Electrodes
Institutions: Unversity of California, San Francisco.
Existing methods for picoinjecting reagents into microfluidic drops require metal electrodes integrated into the microfluidic chip. The integration of these electrodes adds cumbersome and error-prone steps to the device fabrication process. We have developed a technique that obviates the needs for metal electrodes during picoinjection. Instead, it uses the injection fluid itself as an electrode, since most biological reagents contain dissolved electrolytes and are conductive. By eliminating the electrodes, we reduce device fabrication time and complexity, and make the devices more robust. In addition, with our approach, the injection volume depends on the voltage applied to the picoinjection solution; this allows us to rapidly adjust the volume injected by modulating the applied voltage. We demonstrate that our technique is compatible with reagents incorporating common biological compounds, including buffers, enzymes, and nucleic acids.
Bioengineering, Issue 86, Droplet microfluidics, picoinjection, lab on a chip, electrodes, microfabrication
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
Characterization of Electrode Materials for Lithium Ion and Sodium Ion Batteries Using Synchrotron Radiation Techniques
Institutions: Lawrence Berkeley National Laboratory, University of Illinois at Chicago, Stanford Synchrotron Radiation Lightsource, Haldor Topsøe A/S, PolyPlus Battery Company.
Intercalation compounds such as transition metal oxides or phosphates are the most commonly used electrode materials in Li-ion and Na-ion batteries. During insertion or removal of alkali metal ions, the redox states of transition metals in the compounds change and structural transformations such as phase transitions and/or lattice parameter increases or decreases occur. These behaviors in turn determine important characteristics of the batteries such as the potential profiles, rate capabilities, and cycle lives. The extremely bright and tunable x-rays produced by synchrotron radiation allow rapid acquisition of high-resolution data that provide information about these processes. Transformations in the bulk materials, such as phase transitions, can be directly observed using X-ray diffraction (XRD), while X-ray absorption spectroscopy (XAS) gives information about the local electronic and geometric structures (e.g.
changes in redox states and bond lengths). In situ
experiments carried out on operating cells are particularly useful because they allow direct correlation between the electrochemical and structural properties of the materials. These experiments are time-consuming and can be challenging to design due to the reactivity and air-sensitivity of the alkali metal anodes used in the half-cell configurations, and/or the possibility of signal interference from other cell components and hardware. For these reasons, it is appropriate to carry out ex situ
on electrodes harvested from partially charged or cycled cells) in some cases. Here, we present detailed protocols for the preparation of both ex situ
and in situ
samples for experiments involving synchrotron radiation and demonstrate how these experiments are done.
Physics, Issue 81, X-Ray Absorption Spectroscopy, X-Ray Diffraction, inorganic chemistry, electric batteries (applications), energy storage, Electrode materials, Li-ion battery, Na-ion battery, X-ray Absorption Spectroscopy (XAS), in situ X-ray diffraction (XRD)
High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices
Institutions: University of Wisconsin-Madison, University of Wisconsin-Madison.
A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.
Biomedical Engineering, Issue 31, automated, passive pumping, microfluidic device, high speed, high flow rate
Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture
Institutions: University of Pennsylvania .
As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al.
and Sill et al.
for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al.
for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.
Cellular Biology, Issue 32, Electrospinning, Photocrosslinking, Photopatterning, Tissue Engineering, Scaffolds, Biomaterials, Bioengineering
Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media
Institutions: Vanderbilt University, Vanderbilt University, Vanderbilt University, Vanderbilt University, University of Connecticut, University of Connecticut.
Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 μm. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio
bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.
Microbiology, Issue 39, Microfluidic device, bacterial transport, porous media, colloid, biofilm, filtration theory, artificial habitat, micromodel, PDMS, GFP
Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study
Institutions: Virginia Commonwealth University, Virginia Commonwealth University, University Hospital of Geneva.
Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration. Whole grafts and individual layers were analyzed using uniaxial tensile testing, dynamic compliance, suture retention, and burst strength. Compliance results revealed that changes to the middle/medial layer changed overall graft behavior with whole graft compliance values ranging from 0.8 - 2.8 % / 100 mmHg, while uniaxial results demonstrated an average modulus range of 2.0 - 11.8 MPa. Both modulus and compliance data displayed values within the range of native artery. Mathematical modeling was implemented to show how changes in layer stiffness affect the overall circumferential wall stress, and as a design aid to achieve the best mechanical combination of materials. Overall, the results indicated that a graft can be designed to mimic a tri-layered structure by altering layer properties.
Bioengineering, Issue 47, Electrospinning, Vascular Graft, Multilayer, Polycaprolactone, Elastin
Planar and Three-Dimensional Printing of Conductive Inks
Institutions: University of Illinois at Urbana-Champaign, Lawrence Livermore National Laboratory, University Of California Santa Barbara .
Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3
. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 μm). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (< 1 μm) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.
Bioengineering, Issue 58, Direct-write assembly, silver ink, 3D printing, planar, three-dimensional, microelectrodes, flexible electronics, printed electronics
Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases
Institutions: Trinity College Dublin .
Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso
or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural & Functional Biology (MS&FB) Group1-3
. JoVE video articles describing the method are available1,4
The manual approach for setting up in meso
trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso
crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso
robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline.
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso
crystallogenesis. The first robot was developed in the MS&FB Group5,6
. The other two have recently become available and are included here for completeness.
An overview of the protocol covered in this article is presented in Figure 1.
All manipulations were performed at room temperature (~20 °C) under ambient conditions.
Materials Science, Issue 67, automation, crystallization, glass sandwich plates, high-throughput, in meso, lipidic cubic phase, lipidic mesophases, macromolecular X-ray crystallography, membrane protein, receptor, robot
The MultiBac Protein Complex Production Platform at the EMBL
Institutions: EMBL Grenoble Outstation and Unit of Virus Host Cell Interactions (UVHCI) UMR5322.
Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2
Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3
BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4
A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8
The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.
Molecular Biology, Issue 77, Genetics, Bioengineering, Virology, Biochemistry, Microbiology, Basic Protocols, Genomics, Proteomics, Automation, Laboratory, Biotechnology, Multiprotein Complexes, Biological Science Disciplines, Robotics, Protein complexes, multigene delivery, recombinant expression, baculovirus system, MultiBac platform, standard operating procedures (SOP), cell, culture, DNA, RNA, protein, production, sequencing
Development of Whispering Gallery Mode Polymeric Micro-optical Electric Field Sensors
Institutions: Southern Methodist University.
Optical modes of dielectric micro-cavities have received significant attention in recent years for their potential in a broad range of applications. The optical modes are frequently referred to as "whispering gallery modes" (WGM) or "morphology dependent resonances" (MDR) and exhibit high optical quality factors. Some proposed applications of micro-cavity optical resonators are in spectroscopy1
, micro-cavity laser technology2
, optical communications3-6
as well as sensor technology. The WGM-based sensor applications include those in biology7
, trace gas detection8
, and impurity detection in liquids9
. Mechanical sensors based on microsphere resonators have also been proposed, including those for force10,11
and wall shear stress14
. In the present, we demonstrate a WGM-based electric field sensor, which builds on our previous studies15,16
. A candidate application of this sensor is in the detection of neuronal action potential.
The electric field sensor is based on polymeric multi-layered dielectric microspheres. The external electric field induces surface and body forces on the spheres (electrostriction effect) leading to elastic deformation. This change in the morphology of the spheres, leads to shifts in the WGM. The electric field-induced WGM shifts are interrogated by exciting the optical modes of the spheres by laser light. Light from a distributed feedback (DFB) laser (nominal wavelength of ~ 1.3 μm) is side-coupled into the microspheres using a tapered section of a single mode optical fiber. The base material of the spheres is polydimethylsiloxane (PDMS). Three microsphere geometries are used: (1) PDMS sphere with a 60:1 volumetric ratio of base-to-curing agent mixture, (2) multi layer sphere with 60:1 PDMS core, in order to increase the dielectric constant of the sphere, a middle layer of 60:1 PDMS that is mixed with varying amounts (2% to 10% by volume) of barium titanate and an outer layer of 60:1 PDMS and (3) solid silica sphere coated with a thin layer of uncured PDMS base. In each type of sensor, laser light from the tapered fiber is coupled into the outermost layer that provides high optical quality factor WGM (Q ~ 106
). The microspheres are poled for several hours at electric fields of ~ 1 MV/m to increase their sensitivity to electric field.
Mechanical Engineering, Issue 71, Physics, Optics, Materials Science, Chemical Engineering, electrostatics, optical fibers, optical materials, optical waveguides, optics, optoelectronics, photonics, geometrical optics, sensors, electric field, dielectric resonators, micro-spheres, whispering gallery mode, morphology dependent resonance, PDMS
Designing a Bio-responsive Robot from DNA Origami
Institutions: Bar-Ilan University.
Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1
, they can be utilized in parallel computing2,3
, recognize and bind molecular or cellular targets4,5
, catalyze chemical reactions6,7
, and generate calculated responses in a biological system8,9
. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12
, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13
, and several variations on this theme have since been realized using various techniques11,12,14,15
. However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16
. In this technique, the folding of a long (>7,000 bases) single-stranded DNA 'scaffold'
is directed to a desired shape by hundreds of short complementary strands termed 'staples'
. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18
The current paper will focus on the caDNAno 2.0 software19
developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20
. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21
to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.
Bioengineering, Issue 77, Genetics, Biomedical Engineering, Molecular Biology, Medicine, Genomics, Nanotechnology, Nanomedicine, DNA origami, nanorobot, caDNAno, DNA, DNA Origami, nucleic acids, DNA structures, CAD, sequencing
High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay
Institutions: Massachusetts General Hospital.
We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson's disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla
luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla
luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.
Cellular Biology, Issue 88, Luciferases, Gene Transfer Techniques, Transfection, High-Throughput Screening Assays, Transfections, Robotics
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
AC Electrokinetic Phenomena Generated by Microelectrode Structures
Institutions: Drexel University, Drexel University.
The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, which is essential for Lab-on-a-Chip applications. AC electrokinetic phenomena use electric fields to generate forces that act on fluids or suspended particles (including those made of dielectric or biological material) and cause them to move in astonishing ways1, 2
. Within a single channel, AC electrokinetics can accomplish many essential on-chip operations such as active micro-mixing, particle separation, particle positioning and micro-pattering. A single device may accomplish several of those operations by simply adjusting operating parameters such as frequency or amplitude of the applied voltage. Suitable electric fields can be readily created by micro-electrodes integrated into microchannels. It is clear from the tremendous growth in this field that AC electrokinetics will likely have a profound effect on healthcare diagnostics3-5
, environmental monitoring6
and homeland security7
In general, there are three AC Electrokinetic phenomena (AC electroosmosis, dielectrophoresis and AC electrothermal effect) each with unique dependencies on the operating parameters. A change in these operating parameters can cause one phenomena to become dominant over another, thus changing the particle or fluid behavior.
It is difficult to predict the behavior of particles and fluids due to the complicated physics that underlie AC electrokinetics. It is the goal of this publication to explain the physics and elucidate particle and fluid behavior. Our analysis also covers how to fabricate the electrode structures that generate them, and how to interpret a wide number of experimental observations using several popular device designs. This video article will help scientists and engineers understand these phenomena and may encourage them to start using AC Electrokinetics in their research.
Bioengineering, Issue 17, AC Electrokinetics, AC Electroosmosis, Dielectrophoresis, Electrothermal Effect, Microelectrode, Microfluidics, Simulation, Microsphere, Microfabrication
BioMEMS: Forging New Collaborations Between Biologists and Engineers
Institutions: University of California, Irvine (UCI).
This video describes the fabrication and use of a microfluidic device to culture central nervous system (CNS) neurons. This device is compatible with live-cell optical microscopy (DIC and phase contrast), as well as confocal and two photon microscopy approaches. This method uses precision-molded polymer parts to create miniature multi-compartment cell culture with fluidic isolation. The compartments are made of tiny channels with dimensions that are large enough to culture neurons in well-controlled fluidic microenvironments. Neurons can be cultured for 2-3 weeks within the device, after which they can be fixed and stained for immunocytochemistry. Axonal and somal compartments can be maintained fluidically isolated from each other by using a small hydrostatic pressure difference; this feature can be used to localize soluble insults to one compartment for up to 20 h after each medium change. Fluidic isolation enables collection of pure axonal fraction and biochemical analysis by PCR. The microfluidic device provides a highly adaptable platform for neuroscience research and may find applications in modeling CNS injury and neurodegeneration.
Neuroscience, Issue 9, Microfluidics, Bioengineering, Neuron