共查询到20条相似文献,搜索用时 15 毫秒
1.
Masamune Morita Hitoyoshi Yamashita Masayuki Hayakawa Hiroaki Onoe Masahiro Takinoue 《Journal of visualized experiments : JoVE》2016,(108)
Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 µl) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies. 相似文献
2.
The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains. 相似文献
3.
William J. McCarty Ljupcho Prodanov Shyam Sundhar Bale Abhinav Bhushan Rohit Jindal Martin L. Yarmush O. Berk Usta 《Journal of visualized experiments : JoVE》2015,(103)
Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices. 相似文献
4.
Yair Glick Dorit Avrahami Efrat Michaely Doron Gerber 《Journal of visualized experiments : JoVE》2012,(66)
Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology. 相似文献
5.
Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 µm to 50 µm in diameter while using large channels that are robust against fouling and clogging. 相似文献
6.
Olivier Galy Kais Zrelli Patricia Latour-Lambert Lyndsey Kirwan Nelly Henry 《Journal of visualized experiments : JoVE》2014,(87)
Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in these structures are held together by physical interactions mediated by a network of extracellular polymeric substances. Bacterial biofilms impact many human activities and the understanding of their properties is crucial for a better control of their development — maintenance or eradication — depending on their adverse or beneficial outcome. This paper describes a novel methodology aiming to measure in situ the local physical properties of the biofilm that had been, until now, examined only from a macroscopic and homogeneous material perspective. The experiment described here involves introducing magnetic particles into a growing biofilm to seed local probes that can be remotely actuated without disturbing the structural properties of the biofilm. Dedicated magnetic tweezers were developed to exert a defined force on each particle embedded in the biofilm. The setup is mounted on the stage of a microscope to enable the recording of time-lapse images of the particle-pulling period. The particle trajectories are then extracted from the pulling sequence and the local viscoelastic parameters are derived from each particle displacement curve, thereby providing the 3D-spatial distribution of the parameters. Gaining insights into the biofilm mechanical profile is essential from an engineer''s point of view for biofilm control purposes but also from a fundamental perspective to clarify the relationship between the architectural properties and the specific biology of these structures. 相似文献
7.
Erika J. Fong Chao Huang Julie Hamilton William J. Benett Mihail Bora Alison Burklund Thomas R. Metz Maxim Shusteff 《Journal of visualized experiments : JoVE》2015,(105)
A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15–1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing. 相似文献
8.
Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system’s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (>70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial. 相似文献
9.
Simona Bottero Tomas Storck Timo J. Heimovaara Mark C.M. van Loosdrecht Michael V. Enzien 《Biofouling》2013,29(9):1069-1086
A two-dimensional pore-scale numerical model was developed to evaluate the dynamics of preferential flow paths in porous media caused by bioclogging. The liquid flow and solute transport through the pore network were coupled with a biofilm model including biomass attachment, growth, decay, lysis, and detachment. Blocking of all but one flow path was obtained under constant liquid inlet flow rate and biomass detachment caused by shear forces only. The stable flow path formed when biofilm detachment balances growth, even with biomass weakened by decay. However, shear forces combined with biomass lysis upon starvation could produce an intermittently shifting location of flow channels. Dynamic flow pathways may also occur when combined liquid shear and pressure forces act on the biofilm. In spite of repeated clogging and unclogging of interconnected pore spaces, the average permeability reached a quasi-constant value. Oscillations in the medium permeability were more pronounced for weaker biofilms. 相似文献
10.
Carina Monico Gionata Belcastro Francesco Vanzi Francesco S. Pavone Marco Capitanio 《Journal of visualized experiments : JoVE》2014,(90)
The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable. 相似文献
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12.
Maxim Kalashnikov Jennifer Campbell Jean C. Lee Andre Sharon Alexis F. Sauer-Budge 《Journal of visualized experiments : JoVE》2014,(83)
We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step - bacterial growth - is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane (PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment. 相似文献
13.
In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal. 相似文献
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15.
A packed bed biofilm reactor inoculated with pure culture Pseudomonas aeruginosa was run under high substrate loading and constant flow rate conditions. The 3.1-cm-diameter cylindrical reactor was 5 cm in length and packed with 1-mm glass beads. Daily observations of biofilm thickness, influent and effluent glucose substrate concentration, and effluent dissolved and total organic carbon were made during the 13-day experiment. Biofilm thickness appeared to rech quasi-steady-state condition after 10 days. A published biofilm process simulation program (AQUASIM) was used to analyze experimental data. Comparison of observed and simulated variables revealed three distinct phases of biofilm accumulation during the experiment: an initial phase, a growth phase, and a mature biofilm phase. Different combinations of biofilm and mass transport process variables were found to be important during each phase. Biofilm detachment was highly correlated with shear at the biofilm surface during all three phases of biofilm development. (c) 1995 John Wiley & Sons, Inc. 相似文献
16.
Shanshan Wang Encheng Li Yanghui Gao Yan Wang Zhe Guo Jiarui He Jianing Zhang Zhancheng Gao Qi Wang 《PloS one》2013,8(2)
Invadopodia or invasive feet, which are actin-rich membrane protrusions with matrix degradation activity formed by invasive cancer cells, are a key determinant in the malignant invasive progression of tumors and represent an important target for cancer therapies. In this work, we presented a microfluidic 3D culture device with continuous supplement of fresh media via a syringe pump. The device mimicked tumor microenvironment in vivo and could be used to assay invadopodia formation and to study the mechanism of human lung cancer invasion. With this device, we investigated the effects of epidermal growth factor (EGF) and matrix metalloproteinase (MMP) inhibitor, GM6001 on invadopodia formation by human non-small cell lung cancer cell line A549 in 3D matrix model. This device was composed of three units that were capable of achieving the assays on one control group and two experimental groups'' cells, which were simultaneously pretreated with EGF or GM6001 in parallel. Immunofluorescence analysis of invadopodia formation and extracellular matrix degradation was conducted using confocal imaging system. We observed that EGF promoted invadopodia formation by A549 cells in 3D matrix and that GM6001 inhibited the process. These results demonstrated that epidermal growth factor receptor (EGFR) signaling played a significant role in invadopodia formation and related ECM degradation activity. Meanwhile, it was suggested that MMP inhibitor (GM6001) might be a powerful therapeutic agent targeting invadopodia formation in tumor invasion. This work clearly demonstrated that the microfluidic-based 3D culture device provided an applicable platform for elucidating the mechanism of cancer invasion and could be used in testing other anti-invasion agents. 相似文献
17.
Autohydrogenotrophic Denitrifying Microbial Community in a Glass Beads Biofilm Reactor 总被引:1,自引:0,他引:1
A glass bead biofilm reactor was operated using H2 as an electron donor to remove nitrate at 150 mg NO3–N l−1 to below detection level. The microbial community in the glass beads biofilm reactor was investigated by using denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis. In DGGE analysis of the biofilm, five bands were dominant and indicated the presence of eight β-proteobacteria, one γ-proteobacteria and twelve clostridia. An unculturable Hydrogenophaga sp., which is a new genus of hydrogen-oxidizing bacterium was dominant in microbial community of the biofilm reactor. 相似文献
18.
Nathan J. Wittenberg Timothy W. Johnson Luke R. Jordan Xiaohua Xu Arthur E. Warrington Moses Rodriguez Sang-Hyun Oh 《Journal of visualized experiments : JoVE》2014,(87)
Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified. 相似文献
19.
Sarah J. Vogt Alexis B. Sanderlin Joseph D. Seymour Sarah L. Codd 《Biotechnology and bioengineering》2013,110(5):1366-1375
Biofilm growth in porous media is difficult to study non‐invasively due to the opaqueness and heterogeneity of the systems. Magnetic resonance is utilized to non‐invasively study water dynamics within porous media. Displacement‐relaxation correlation experiments were performed on fluid flow during biofilm growth in a model porous media of mono‐dispersed polystyrene beads. The spin–spin T2 magnetic relaxation distinguishes between the biofilm phase and bulk fluid phase due to water–biopolymer interactions present in the biofilm, and the flow dynamics are measured using PGSE NMR experiments. By correlating these two measurements, the effects of biofilm growth on the fluid dynamics can be separated into a detailed analysis of both the biofilm phase and the fluid phase simultaneously within the same experiment. Within the displacement resolution of these experiments, no convective flow was measured through the biomass. An increased amount of longitudinal hydrodynamic dispersion indicates increased hydrodynamic mixing due to fluid channeling caused by biofilm growth. The effect of different biofilm growth conditions was measured by varying the strength of the bacterial growth medium. Biotechnol. Bioeng. 2013; 110: 1366–1375. © 2012 Wiley Periodicals, Inc. 相似文献
20.
Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms. 相似文献