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Sample records for microfluidic protein patterning

  1. Microfluidic Tools for Protein Crystallography

    NASA Astrophysics Data System (ADS)

    Abdallah, Bahige G.

    X-ray crystallography is the most widely used method to determine the structure of proteins, providing an understanding of their functions in all aspects of life to advance applications in fields such as drug development and renewable energy. New techniques, namely serial femtosecond crystallography (SFX), have unlocked the ability to unravel the structures of complex proteins with vital biological functions. A key step and major bottleneck of structure determination is protein crystallization, which is very arduous due to the complexity of proteins and their natural environments. Furthermore, crystal characteristics govern data quality, thus need to be optimized to attain the most accurate reconstruction of the protein structure. Crystal size is one such characteristic in which narrowed distributions with a small modal size can significantly reduce the amount of protein needed for SFX. A novel microfluidic sorting platform was developed to isolate viable ~200 nm -- ~600 nm photosystem I (PSI) membrane protein crystals from ~200 nm -- ~20 ?m crystal samples using dielectrophoresis, as confirmed by fluorescence microscopy, second-order nonlinear imaging of chiral crystals (SONICC), and dynamic light scattering. The platform was scaled-up to rapidly provide 100s of microliters of sorted crystals necessary for SFX, in which similar crystal size distributions were attained. Transmission electron microscopy was used to view the PSI crystal lattice, which remained well-ordered postsorting, and SFX diffraction data was obtained, confirming a high-quality, viable crystal sample. Simulations indicated sorted samples provided accurate, complete SFX datasets with 3500-fold less protein than unsorted samples. Microfluidic devices were also developed for versatile, rapid protein crystallization screening using nanovolumes of sample. Concentration gradients of protein and precipitant were generated to crystallize PSI, phycocyanin, and lysozyme using modified counterdiffusion

  2. Patterned Plasmonic Nanoparticle Arrays for Microfluidic and Multiplexed Biological Assays.

    PubMed

    He, Jie; Boegli, Michelle; Bruzas, Ian; Lum, William; Sagle, Laura

    2015-11-17

    For applications ranging from medical diagnostics and drug screening to chemical and biological warfare detection, inexpensive, rapid-readout, portable devices are required. Localized surface plasmon resonance (LSPR) technologies show substantial promise toward meeting these goals, but the generation of portable, multiplexed and/or microfluidic devices incorporating sensitive nanoparticle arrays is only in its infancy. Herein, we have combined photolithography with Hole Mask Colloidal lithography to pattern uniform nanoparticle arrays for both microfluidic and multiplexed devices. The first proof-of-concept study is carried out with 5- and 7-channel microfluidic devices to acquire one-shot binding curves and protein binding kinetic data. The second proof-of-concept study involved the fabrication of a 96-spot plate that can be inserted into a standard plate reader for the multiplexed detection of protein binding. This versatile fabrication technique should prove useful in next generation chips for bioassays and genetic screening. PMID:26494412

  3. Rapid Protein Separations in Microfluidic Devices

    NASA Technical Reports Server (NTRS)

    Fan, Z. H.; Das, Champak; Xia, Zheng; Stoyanov, Alexander V.; Fredrickson, Carl K.

    2004-01-01

    This paper describes fabrication of glass and plastic microfluidic devices for protein separations. Although the long-term goal is to develop a microfluidic device for two-dimensional gel electrophoresis, this paper focuses on the first dimension-isoelectric focusing (IEF). A laser-induced fluorescence (LIF) imaging system has been built for imaging an entire channel in an IEF device. The whole-channel imaging eliminates the need to migrate focused protein bands, which is required if a single-point detector is used. Using the devices and the imaging system, we are able to perform IEF separations of proteins within minutes rather than hours in traditional bench-top instruments.

  4. Surface patterning of bonded microfluidic channels

    PubMed Central

    Priest, Craig

    2010-01-01

    Microfluidic channels in which multiple chemical and biological processes can be integrated into a single chip have provided a suitable platform for high throughput screening, chemical synthesis, detection, and alike. These microchips generally exhibit a homogeneous surface chemistry, which limits their functionality. Localized surface modification of microchannels can be challenging due to the nonplanar geometries involved. However, chip bonding remains the main hurdle, with many methods involving thermal or plasma treatment that, in most cases, neutralizes the desired chemical functionality. Postbonding modification of microchannels is subject to many limitations, some of which have been recently overcome. Novel techniques include solution-based modification using laminar or capillary flow, while conventional techniques such as photolithography remain popular. Nonetheless, new methods, including localized microplasma treatment, are emerging as effective postbonding alternatives. This Review focuses on postbonding methods for surface patterning of microchannels. PMID:21045927

  5. Protein immobilization techniques for microfluidic assays

    PubMed Central

    Kim, Dohyun; Herr, Amy E.

    2013-01-01

    Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization. PMID:24003344

  6. Microfluidic DNA extraction using a patterned aluminum oxide membrane

    NASA Astrophysics Data System (ADS)

    Kim, Jungkyu; Gale, Bruce K.

    2006-01-01

    A DNA extraction system was designed and fabricated using an AOM (aluminum oxide membrane) with 200 nm pores and PDMS microfluidic channels. The membrane was patterned using soft lithography techniques and SU-8 photolithography on the membrane. After making the pattern with SU-8, the AOM was observed using an SEM (scanning electro microscope) to verify the AOM structure was not damaged. From the SEM images, the AOM structure was not different after modification with SU-8. To complete the system, a PDMS mold for the microfluidic channels was made by soft lithography. Using the SU-8 mold, PDMS microchannels were cast using PDMS with a low polymer to curing agent ratio to provide adhesion between the patterned membrane and microfluidic channel. Then, the patterned membrane was sandwiched between PDMS microfluidic channels in a parallel format. The completed system was tested with 10ug of Lambda DNA mixed with the fluorescent dye SYBR Green I. Following DNA extraction, the surface of each well was examined with fluorescence microscopy while embedded in the microfluidic system. Extracted and immobilized DNA on the AOM was observed in almost every separation well. This microsystem, referred to as a membrane-on-a-chip, has potential applications in high-throughput DNA extraction and analysis, with the possibility of being integrated into polymer-based microfluidic systems.

  7. Characterization of soy protein nanoparticles prepared by high shear microfluidization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soy protein nanoparticles were produced with a microfluidizer and characterized in terms of particle size, size distribution, morphology, rheological properties, and aggregate structure. Three stages of structure breakdown were observed when the soy protein dispersion was passed through the microflu...

  8. Structural characterization of soy protein nanoparticles from high shear microfluidization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soy protein nanoparticles were produced with a microfluidizer and characterized in terms of particle size, size distribution, morphology, rheological properties, and aggregate structure. Three stages of structure breakdown were observed when the soy protein dispersion was passed through the microflu...

  9. Digital microfluidic assay for protein detection.

    PubMed

    Mok, Janine; Mindrinos, Michael N; Davis, Ronald W; Javanmard, Mehdi

    2014-02-11

    Global studies of the human proteome have revealed a plethora of putative protein biomarkers. However, their application for early disease detection remains at a standstill without suitable methods to realize their utility in the clinical setting. There thus continues to be tremendous interest in developing new technology for sensitive protein detection that is both low in cost and carries a small footprint to be able to be used at the point of care. The current gold standard method for protein biomarker detection is the ELISA, which measures protein abundance using bulky fluorescent scanners that lack portability. Here, we present a digital microfluidic platform for protein biomarker detection that is low in cost compared with standard optical detection methods, without any compromise in sensitivity. This platform furthermore makes use of simple electronics, enabling its translation into a portable handheld device, and has been developed in a manner that can easily be adapted to assay different types of proteomic biomarkers. We demonstrate its utility in quantifying not only protein abundance, but also activity. Interleukin-6 abundance could be assayed from concentrations as low as 50 pM (an order of magnitude lower than that detectable by a comparable laboratory designed ELISA) using less than 5 μL of sample, and Abelson tyrosine kinase activity was detectable in samples containing 100 pM of kinase. PMID:24449893

  10. Microfluidic Mixers for Studying Protein Folding

    PubMed Central

    Waldauer, Steven A.; Wu, Ling; Yao, Shuhuai; Bakajin, Olgica; Lapidus, Lisa J.

    2012-01-01

    The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 μs2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4. Laminar flow is achieved for Reynolds numbers Re ≤100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 μm wide and 10 μm deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 μm in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 μL/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 μM (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM. The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 μm in 1 ms

  11. Patterned Immobilization of Antibodies within Roll-to-Roll Hot Embossed Polymeric Microfluidic Channels

    PubMed Central

    Feyssa, Belachew; Liedert, Christina; Kivimaki, Liisa; Johansson, Leena-Sisko; Jantunen, Heli; Hakalahti, Leena

    2013-01-01

    This paper describes a method for the patterned immobilization of capture antibodies into a microfluidic platform fabricated by roll-to-roll (R2R) hot embossing on poly (methyl methacrylate) (PMMA). Covalent attachment of antibodies was achieved by two sequential inkjet printing steps. First, a polyethyleneimine (PEI) layer was deposited onto oxygen plasma activated PMMA foil and further cross-linked with glutaraldehyde (GA) to provide an amine-reactive aldehyde surface (PEI-GA). This step was followed by a second deposition of antibody by overprinting on the PEI-GA patterned PMMA foil. The PEI polymer ink was first formulated to ensure stable drop formation in inkjet printing and the printed films were characterized using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Anti-CRP antibody was patterned on PMMA foil by the developed method and bonded permanently with R2R hot embossed PMMA microchannels by solvent bonding lamination. The functionality of the immobilized antibody inside the microfluidic channel was evaluated by fluorescence-based sandwich immunoassay for detection of C-reactive protein (CRP). The antibody-antigen assay exhibited a good level of linearity over the range of 10 ng/ml to 500 ng/ml (R2 = 0.991) with a calculated detection limit of 5.2 ng/ml. The developed patterning method is straightforward, rapid and provides a versatile approach for creating multiple protein patterns in a single microfluidic channel for multiplexed immunoassays. PMID:23874811

  12. Multiplexed microfluidic quantification of proteins in serum

    NASA Astrophysics Data System (ADS)

    Rajan, Nitin; Rajauria, Sukumar; Cleland, Andrew

    2015-03-01

    Rapid and low cost immunoassays targeting proteins in blood or other bodily fluids are highly sought after for point-of-care devices and early screening of patients. Immunoturbidimetric assays utilize latex particles functionalized with antibodies, with particle aggregation in the presence of the analyte detected by a change in absorbance. Using a high throughput micro-fluidic particle analyzer based solely on electrical signals (resistive pulse sensing), we are able to accurately quantify the degree of aggregation by analyzing the changes in the particle size distribution. Thus we study the aggregation of streptavidin (SAv) coated beads in the presence of biotinylated bovine serum albumin as a proof-of-principle assay and extract the binding capacity of the SAv beads from the dose-response curve. We also use our aggregation measurement platform to characterize a commercial C-reactive protein (CRP) immunoturbidimetric assay (hsCRP, Diazyme Inc.). We obtain a linear calibration curve as well as a better limit of detection of CRP than that obtained by absorbance measurements. By using different bead sizes functionalized with different antibodies, multiplexed analyte detection is also possible. We demonstrate this by combining the commercial anti-CRP functionalized beads (0.4 microns) with biotin coated beads (1.0 microns), and carry out the simultaneous detection of SAv and CRP in a single sample.

  13. Functional polymer sheet patterning using microfluidics.

    PubMed

    Li, Minggan; Humayun, Mouhita; Kozinski, Janusz A; Hwang, Dae Kun

    2014-07-22

    Poly(dimethylsiloxane) (PDMS)-based microfluidics provide a novel approach to advanced material synthesis. While PDMS has been successfully used in a wide range of industrial applications, due to the weak mechanical property channels generally possess low aspect ratios (AR) and thus produce microparticles with similarly low ARs. By increasing the channel width to nearly 1 cm, AR to 267, and implementing flow lithography, we were able to establish the slit-channel lithography. Not only does this allow us to synthesize sheet materials bearing multiscale features and tunable chemical anisotropy but it also allows us to fabricate functional layered sheet structures in a one-step, high-throughput fashion. We showcased the technique's potential role in various applications, such as the synthesis of planar material with micro- and nanoscale features, surface morphologies, construction of tubular and 3D layered hydrogel tissue scaffolds, and one-step formation of radio frequency identification (RFID) tags. The method introduced offers a novel route to functional sheet material synthesis and sheet system fabrication. PMID:24967616

  14. Protein Microarrays with Novel Microfluidic Methods: Current Advances

    PubMed Central

    Dixit, Chandra K.; Aguirre, Gerson R.

    2014-01-01

    Microfluidic-based micromosaic technology has allowed the pattering of recognition elements in restricted micrometer scale areas with high precision. This controlled patterning enabled the development of highly multiplexed arrays multiple analyte detection. This arraying technology was first introduced in the beginning of 2001 and holds tremendous potential to revolutionize microarray development and analyte detection. Later, several microfluidic methods were developed for microarray application. In this review we discuss these novel methods and approaches which leverage the property of microfluidic technologies to significantly improve various physical aspects of microarray technology, such as enhanced imprinting homogeneity, stability of the immobilized biomolecules, decreasing assay times, and reduction of the costs and of the bulky instrumentation.

  15. High throughput and multiplex localization of proteins and cells for in situ micropatterning using pneumatic microfluidics.

    PubMed

    Wang, Jian-Chun; Liu, Wenming; Tu, Qin; Ma, Chao; Zhao, Lei; Wang, Yaolei; Ouyang, Jia; Pang, Long; Wang, Jinyi

    2015-02-01

    Micropatterning technologies are emerging as an enabling tool for various microfluidic-based applications in life sciences. However, the high throughput and multiplex localization of multiple bio-components in a microfluidic device has not yet been well established. In this paper, we describe a simple and in situ micropatterning method using an integrated microfluidic device with pneumatic microstructures (PμSs) for highly controllable immobilization of both proteins and cells in a high throughput, geometry-dynamic, and multi-patterning way. The precise Pluronic F127 passivation of a microchamber surface except the PμS-blocked regions was performed and characterized, and the spatial dynamics and consistency of both the PμSs and protein/cell micropatterning were optically evaluated and quantitatively demonstrated too. Furthermore, a systematic investigation of PμS-assisted micropatterning in microfluidics was carried out. The feature of high throughput and spatial control of micropatterning can be simply realized by using the well-designed PμS arrays. Meanwhile, the co-micropatterning of different proteins (bovine serum albumin and chicken egg albumin) and cells (human umbilical vein endothelial cells and human hepatocellular carcinoma cells) in a microfluidic device was successfully accomplished with the orderly serial manipulation of PμS groups. We demonstrate that PμS-assisted micropatterning can be applied as a convenient microfluidic component for large-scale and diversified protein/cell patterning and manipulation, which could be useful for cell-based tissue organization, high-throughput imaging, protein-related interactions and immunoassays. PMID:25453039

  16. Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices.

    PubMed

    Kalish, Brent; Tsutsui, Hideaki

    2016-01-01

    We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive. PMID:27077551

  17. Hydrogel microfluidics for the patterning of pluripotent stem cells

    NASA Astrophysics Data System (ADS)

    Cosson, S.; Lutolf, M. P.

    2014-03-01

    Biomolecular signaling is of utmost importance in governing many biological processes such as the patterning of the developing embryo where biomolecules regulate key cell-fate decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion. Although state-of-the-art microfluidic technologies allow precise biomolecule delivery in time and space, long-term (stem) cell culture at the micro-scale is often far from ideal due to medium evaporation, limited space for cell growth or shear stress. To overcome these challenges, we here introduce a concept based on hydrogel microfluidics for decoupling conventional, macro-scale cell culture from precise biomolecule delivery through a gel layer. We demonstrate the spatiotemporally controlled neuronal commitment of mouse embryonic stem cells via delivery of retinoic acid gradients. This technique should be useful for testing the effect of dose and timing of biomolecules, singly or in combination, on stem cell fate.

  18. Screening for Host Factors Directly Interacting with RSV Protein: Microfluidics.

    PubMed

    Kipper, Sarit; Avrahami, Dorit; Bajorek, Monika; Gerber, Doron

    2016-01-01

    We present a high-throughput microfluidics platform to identify novel host cell binding partners of respiratory syncytial virus (RSV) matrix (M) protein. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed custom-made gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for binding to RSV M protein.Even small viral proteome, such as that of RSV, presents a challenge due to the fact that viral proteins are usually multifunctional and thus their interaction with the host is complex. Protein microarrays technology allows the interrogation of protein-protein interactions, which could possibly overcome obstacles by using conventional high throughput methods. Using microfluidics platform we have identified new host interactors of M involved in various cellular pathways. A number of microfluidics based assays have already provided novel insights into the virus-host interactome, and the results have important implications for future antiviral strategies aimed at targets of viral protein interactions with the host. PMID:27464694

  19. Cell-free protein synthesis in microfluidic array devices.

    PubMed

    Mei, Qian; Fredrickson, Carl K; Simon, Andrew; Khnouf, Ruba; Fan, Z Hugh

    2007-01-01

    We report the development of a microfluidic array device for continuous-exchange, cell-free protein synthesis. The advantages of protein expression in the microfluidic array include (1) the potential to achieve high-throughput protein expression, matching the throughput of gene discovery; (2) more than 2 orders of magnitude reduction in reagent consumption, decreasing the cost of protein synthesis; and (3) the possibility to integrate with detection for rapid protein analysis, eliminating the need to harvest proteins. The device consists of an array of units, and each unit can be used for production of an individual protein. The unit comprises a tray chamber for in vitro protein expression and a well chamber as a nutrient reservoir. The tray is nested in the well, and they are separated by a dialysis membrane and connected through a microfluidic connection that provides a means to supply nutrients and remove the reaction byproducts. The device is demonstrated by synthesis of green fluorescent protein, chloramphenicol acetyl-transferase, and luciferase. Protein expression in the device lasts 5-10 times longer and the production yield is 13-22 times higher than in a microcentrifuge tube. In addition, we studied the effects of the operation temperature and hydrostatic flow on the protein production yield. PMID:17924644

  20. Pattern Formation Exhibited by Biofilm Formation within Microfluidic Chambers

    PubMed Central

    Cogan, N.G.; Donahue, M.R.; Whidden, Mark; De La Fuente, Leonardo

    2013-01-01

    This article investigates the dynamics of an important bacterial pathogen, Xylella fastidiosa, within artificial plant xylem. The bacterium is the causative agent of a variety of diseases that strike fruit-bearing plants including Pierce’s disease of grapevine. Biofilm colonization within microfluidic chambers was visualized in a laboratory setting, showing robust, regular spatial patterning. We also develop a mathematical model, based on a multiphase approach that is able to capture the spacing of the pattern and points to the role of the exopolymeric substance as the main source of control of the pattern dynamics. We concentrate on estimating the attachment/detachment processes within the chamber because these are two mechanisms that have the potential to be engineered by applying various chemicals to prevent or treat the disease. PMID:23663829

  1. Pattern formation exhibited by biofilm formation within microfluidic chambers.

    PubMed

    Cogan, N G; Donahue, M R; Whidden, Mark; De La Fuente, Leonardo

    2013-05-01

    This article investigates the dynamics of an important bacterial pathogen, Xylella fastidiosa, within artificial plant xylem. The bacterium is the causative agent of a variety of diseases that strike fruit-bearing plants including Pierce's disease of grapevine. Biofilm colonization within microfluidic chambers was visualized in a laboratory setting, showing robust, regular spatial patterning. We also develop a mathematical model, based on a multiphase approach that is able to capture the spacing of the pattern and points to the role of the exopolymeric substance as the main source of control of the pattern dynamics. We concentrate on estimating the attachment/detachment processes within the chamber because these are two mechanisms that have the potential to be engineered by applying various chemicals to prevent or treat the disease. PMID:23663829

  2. Protein Crystal Growth With the Aid of Microfluidics

    NASA Technical Reports Server (NTRS)

    vanderWoerd, Mark

    2003-01-01

    Protein crystallography is one of three well-known methods to obtain the structure of proteins. A major rate limiting step in protein crystallography is protein crystal nucleation and growth, which is still largely a process conducted by trial-and-error methods. Many attempts have been made to improve protein crystal growth by performing growth in microgravity. Although the use of microgravity appears to improve crystal quality in some attempts, this method has been inefficient because several reasons: we lack a fundamental understanding of macromolecular crystal growth in general and of the influence of microgravity in particular, we have to start with crystal growth conditions in microgravity based on conditions on the ground and finally the hardware does not allow for experimental iteration without reloading samples on the ground. To partially accommodate the disadvantages of the current hardware, we have used microfluidic technology (Lab-on-a-Chip devices) to design the concept of a more efficient crystallization device, suitable for use on the International Space Station and in high-throughput applications on the ground. The concept and properties of microfluidics, the application design process, and the advances in protein crystal growth hardware will be discussed in this presentation. Some examples of proteins crystallized in the new hardware will be discussed, including the differences between conventional crystallization versus crystallization in microfluidics.

  3. Patterned Photonic Nitrocellulose for Pseudo-Paper Microfluidics.

    PubMed

    Gao, Bingbing; Liu, Hong; Gu, Zhongze

    2016-05-17

    We report a pseudo-paper microfluidic chip based on patterned photonic nitrocellulose. The photonic nitrocellulose is fabricated using self-assembled monodisperse SiO2 nanoparticles as template. The SiO2 nanoparticles form a photonic crystal having a close-packed hexagonal structure in the microchannels, so the resulting nitrocellulose has a complementary inverse-opal structure. After lamination, a hollow channel is obtained that is partially filled with the photonic nitrocellulose. Owing to the highly ordered photonic structure of the pseudo-paper chip, the flow profile of aqueous solution wicking through the channel is more uniform than conventional paper microfluidic chip. It is also found that the wicking rate of aqueous solution can be easily manipulated by changing the diameter of the self-assembled monodisperse SiO2 nanoparticles, which determines the pore size of the photonic nitrocellulose. The fluorescent enhancement property of the photonic nitrocellulose is used to increase the fluorescent intensity for multiplex detection of two cancer biomarkers. Label-free detection of human immunoglobin G based on the structure color of the photonic nitrocellulose is also demonstrated. PMID:27088587

  4. Modular microfluidics for point-of-care protein purifications

    SciTech Connect

    Millet, L. J.; Lucheon, J. D.; Standaert, R. F.; Retterer, S. T.; Doktycz, M. J.

    2015-01-01

    Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured to suit a variety of fluidic operations or biochemical processes. In conclusion, we demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.

  5. Modular microfluidics for point-of-care protein purifications.

    PubMed

    Millet, L J; Lucheon, J D; Standaert, R F; Retterer, S T; Doktycz, M J

    2015-04-21

    Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured to suit a variety of fluidic operations or biochemical processes. We demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules. PMID:25740172

  6. Microfluidic IEF technique for sequential phosphorylation analysis of protein kinases

    NASA Astrophysics Data System (ADS)

    Choi, Nakchul; Song, Simon; Choi, Hoseok; Lim, Bu-Taek; Kim, Young-Pil

    2015-11-01

    Sequential phosphorylation of protein kinases play the important role in signal transduction, protein regulation, and metabolism in living cells. The analysis of these phosphorylation cascades will provide new insights into their physiological functions in many biological functions. Unfortunately, the existing methods are limited to analyze the cascade activity. Therefore, we suggest a microfluidic isoelectric focusing technique (μIEF) for the analysis of the cascade activity. Using the technique, we show that the sequential phosphorylation of a peptide by two different kinases can be successfully detected on a microfluidic chip. In addition, the inhibition assay for kinase activity and the analysis on a real sample have also been conducted. The results indicate that μIEF is an excellent means for studies on phosphorylation cascade activity.

  7. Magnetoactive Sponges for Dynamic Control of Microfluidic Flow Patterns in Microphysiological Systems

    PubMed Central

    Yen, Ringo; Chan, Hon Fai; Leong, Kam W; Truskey, George A; Zhao, Xuanhe

    2014-01-01

    We developed a microfluidic flow-control system capable of dynamically generating various flow patterns on demand. The flow-control system is based on novel magnetoactive sponges embedded in microfluidic flow channels. Applying a non-uniform magnetic field compresses the magnetoactive sponge, significantly reducing porosity and hydraulic conductivity. Tuning the applied magnetic field can dynamically vary the flow rate in the microfluidic channel. Pulsatile and physiological flow patterns with frequency between 1 and 3 Hz, flow rates between 0.5 and 10 μL/min and duration over 3 weeks have been achieved. Smooth muscle cells in engineered blood vessels perfused for 7 days aligned perpendicular to the flow direction under pulsatile but not steady flow, similar to the in vivo orientation. Owing to its various advantages over traditional flow-control methods, the new system potentially has important applications in microfluidic-based microphysiological systems to simulate the physiological nature of blood flow. PMID:24310854

  8. Microsecond Microfluidic Mixing for Investigation of Protein Folding Kinetics

    SciTech Connect

    Hertzog, D E; Santiago, J G; Bakajin, O

    2003-06-25

    We have developed and characterized a mixer to study the reaction kinetics of protein folding on a microsecond timescale. The mixer uses hydrodynamic focusing of pressure-driven flow in a microfluidic channel to reduce diffusion times as first demonstrated by Knight et al.[1]. Features of the mixer include 1 {micro}s mixing times, sample consumptions of order 1 nl/s, loading sample volumes on the order of microliters, and the ability to manufacture in fused silica for compatibility with most spectroscopic methods.

  9. Microsecond Microfluidic Mixing for Investigation of Protein Folding Kinetics

    SciTech Connect

    Hertzog, D E; Santiago, J G; Bakajin, O

    2005-02-10

    We have developed and characterized a mixer to study the reaction kinetics of protein folding on a microsecond timescale. The mixer uses hydrodynamic focusing of pressure-driven flow in a microfluidic channel to reduce diffusion times as first demonstrated by Knight et al.[1]. Features of the mixer include 1 {micro}s mixing times, sample consumptions of order 1 nl/s, loading sample volumes on the order of microliters, and the ability to manufacture in fused silica for compatibility with most spectroscopic methods.

  10. Effect of microfluidized and stearic acid modified soy protein in natural rubber

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microfluidized and stearic acid modified soy protein aggregates were used to reinforced natural rubber. The size of soy protein particles was reduced with a microfluidizing and ball milling process. Filler size reduction with longer ball milling time tends to increase tensile strength of the rubber ...

  11. Laminated microfluidic system for small sample protein analysis

    PubMed Central

    Saedinia, Sara; Nastiuk, Kent L.; Krolewski, John J.; Li, G. P.; Bachman, Mark

    2014-01-01

    We describe a technology based on lamination that allows for the production of highly integrated 3D devices suitable for performing a wide variety of microfluidic assays. This approach uses a suite of microfluidic coupons (“microfloupons”) that are intended to be stacked as needed to produce an assay of interest. Microfloupons may be manufactured in paper, plastic, gels, or other materials, in advance, by different manufacturers, then assembled by the assay designer as needed. To demonstrate this approach, we designed, assembled, and characterized a microfloupon device that performs sodium-dodecyl-sulfate polyacrylamide gel electrophoresis on a small sample of protein. This device allowed for the manipulation and transport of small amounts of protein sample, tight injection into a thin polyacrylamide gel, electrophoretic separation of the proteins into bands, and subsequent removal of the gel from the device for imaging and further analysis. The microfloupons are rugged enough to handle and can be easily aligned and laminated, allowing for a variety of different assays to be designed and configured by selecting appropriate microfloupons. This approach provides a convenient way to perform assays that have multiple steps, relieving the need to design highly sophisticated devices that incorporate all functions in a single unit, while still achieving the benefits of small sample size, automation, and high speed operation. PMID:24753728

  12. A microfluidic electrochemiluminescent device for detecting cancer biomarker proteins.

    PubMed

    Sardesai, Naimish P; Kadimisetty, Karteek; Faria, Ronaldo; Rusling, James F

    2013-04-01

    We describe an electrochemiluminescence (ECL) immunoarray incorporated into a prototype microfluidic device for highly sensitive protein detection and apply this system to accurate, sensitive measurements of prostate-specific antigen (PSA) and interleukin-6 (IL-6) in serum. The microfluidic system employed three molded polydimethylsiloxane (PDMS) channels on a conductive pyrolytic graphite chip (2.5 × 2.5 cm) inserted into a machined chamber and interfaced with a pump, switching valve, and sample injector. Each of the three PDMS channels encompasses three 3 μL analytical wells. Capture-antibody-decorated single-wall carbon nanotube forests are fabricated in the bottom of the wells. The antigen is captured by these antibodies on the well bottoms. Then, a RuBPY-silica-secondary antibody (Ab2) label is injected to bind to antigen on the array, followed by injection of sacrificial reductant tripropylamine (TPrA) to produce ECL. For detection, the chip is placed into an open-top ECL measuring cell, and the channels are in contact with electrolyte in the chamber. Potential applied at 0.95 V versus Ag/AgCl oxidizes TPrA to produce ECL by redox cycling the RuBPY species in the particles, and ECL light is measured by a charge-coupled device camera. This approach achieved ultralow detection limits of 100 fg mL(-1) for PSA (9 zeptomole) and 10 fg mL(-1) (1 zeptomole) for IL-6 in calf serum, a 10-25-fold improvement of a similar non-microfluidic array. PSA and IL-6 in synthetic cancer patient serum samples were detected in 1.1 h and results correlated well with single-protein enzyme-linked immunosorbent assays. PMID:23307128

  13. A microfluidic electrochemiluminescent device for detecting cancer biomarker proteins

    PubMed Central

    Sardesai, Naimish P.; Kadimisetty, Karteek; Faria, Ronaldo; Rusling, James F.

    2013-01-01

    We describe an electrochemiluminescence (ECL) immunoarray incorporated into a prototype microfluidic device for highly sensitive protein detection, and apply this system to accurate, sensitive measurements of prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum. The microfluidic system employed three molded polydimethylsiloxane (PDMS) channels on a conductive pyrolytic graphite chip (PG) (2.5 × 2.5 cm) inserted into a machined chamber and interfaced with a pump, switching valve and sample injector. Each of the three PDMS channels encompasses three 3 μL analytical wells. Capture-antibody-decorated single-wall carbon nanotube (SWCNT) forests are fabricated in the bottom of the wells. The antigen is captured by these antibodies on the well bottoms. Then a RuBPY-silica-secondary antibody (Ab2) label is injected to bind to antigen on the array, followed by injection of sacrificial reductant tripropylamine (TPrA) to produce ECL. For detection, the chip is placed into an open-top ECL measuring cell, and the channels are in contact with electrolyte in the chamber. Potential applied at 0.95 V vs. SCE oxidizes TPrA to produce ECL by redox cycling the RuBPY species in the particles, and ECL light is measured by a CCD camera. This approach achieved ultralow detection limits (DL) of 100 fg mL-1 for PSA (9 zeptomol) and 10 fg mL-1 (1 zeptomol) for IL-6 in calf serum, a 10-25 fold improvement of a similar non-microfluidic array. PSA and IL-6 in synthetic cancer patient serum samples were detected in 1.1 h and results correlated well with single-protein ELISAs. PMID:23307128

  14. Modular microfluidics for point-of-care protein purifications

    DOE PAGESBeta

    Millet, L. J.; Lucheon, J. D.; Standaert, R. F.; Retterer, S. T.; Doktycz, M. J.

    2015-01-01

    Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured tomore » suit a variety of fluidic operations or biochemical processes. In conclusion, we demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.« less

  15. Soft lithographic patterning of supported lipid bilayers onto a surface and inside microfluidic channels.

    PubMed

    Kim, Pilnam; Lee, Sang Eun; Jung, Ho Sup; Lee, Hea Yeon; Kawai, Tomoji; Suh, Kahp Y

    2006-01-01

    We present simple soft lithographic methods for patterning supported lipid bilayer (SLB) membranes onto a surface and inside microfluidic channels. Micropatterns of polyethylene glycol (PEG)-based polymers were fabricated on glass substrates by microcontact printing or capillary moulding. The patterned PEG surfaces have shown 97 +/- 0.5% reduction in lipid adsorption onto two dimensional surfaces and 95 +/- 1.2% reduction inside microfluidic channels in comparison to glass control. Atomic force microscopy measurements indicated that the deposition of lipid vesicles led to the formation of SLB membranes by vesicle fusion due to hydrophilic interactions with the exposed substrate. Furthermore, the functionality of the patterned SLBs was tested by measuring the binding interactions between biotin (ligand)-labeled lipid bilayer and streptavidin (receptor). SLB arrays were fabricated with spatial resolution down to approximately 500 nm on flat substrate and approximately 1 microm inside microfluidic channels, respectively. PMID:16372069

  16. Development of Microfluidic Systems Enabling High-Throughput Single-Cell Protein Characterization.

    PubMed

    Fan, Beiyuan; Li, Xiufeng; Chen, Deyong; Peng, Hongshang; Wang, Junbo; Chen, Jian

    2016-01-01

    This article reviews recent developments in microfluidic systems enabling high-throughput characterization of single-cell proteins. Four key perspectives of microfluidic platforms are included in this review: (1) microfluidic fluorescent flow cytometry; (2) droplet based microfluidic flow cytometry; (3) large-array micro wells (microengraving); and (4) large-array micro chambers (barcode microchips). We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on three key performance parameters (absolute quantification, sensitivity, and throughput). PMID:26891303

  17. Development of Microfluidic Systems Enabling High-Throughput Single-Cell Protein Characterization

    PubMed Central

    Fan, Beiyuan; Li, Xiufeng; Chen, Deyong; Peng, Hongshang; Wang, Junbo; Chen, Jian

    2016-01-01

    This article reviews recent developments in microfluidic systems enabling high-throughput characterization of single-cell proteins. Four key perspectives of microfluidic platforms are included in this review: (1) microfluidic fluorescent flow cytometry; (2) droplet based microfluidic flow cytometry; (3) large-array micro wells (microengraving); and (4) large-array micro chambers (barcode microchips). We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on three key performance parameters (absolute quantification, sensitivity, and throughput). PMID:26891303

  18. Development of a multiplexed microfluidic proteomic reactor and its application for studying protein-protein interactions.

    PubMed

    Tian, Ruijun; Hoa, Xuyen Dai; Lambert, Jean-Philippe; Pezacki, John Paul; Veres, Teodor; Figeys, Daniel

    2011-06-01

    Mass spectrometry-based proteomics techniques have been very successful for the identification and study of protein-protein interactions. Typically, immunopurification of protein complexes is conducted, followed by protein separation by gel electrophoresis and in-gel protein digestion, and finally, mass spectrometry is performed to identify the interacting partners. However, the manual processing of the samples is time-consuming and error-prone. Here, we developed a polymer-based microfluidic proteomic reactor aimed at the parallel analysis of minute amounts of protein samples obtained from immunoprecipitation. The design of the proteomic reactor allows for the simultaneous processing of multiple samples on the same devices. Each proteomic reactor on the device consists of SCX beads packed and restricted into a 1 cm microchannel by two integrated pillar frits. The device is fabricated using a combination of low-cost hard cyclic olefin copolymer thermoplastic and elastomeric thermoplastic materials (styrene/(ethylene/butylenes)/styrene) using rapid hot-embossing replication techniques with a polymer-based stamp. Three immunopurified protein samples are simultaneously captured, reduced, alkylated, and digested on the device within 2-3 h instead of the days required for the conventional protein-protein interaction studies. The limit of detection of the microfluidic proteomic reactor was shown to be lower than 2 ng of protein. Furthermore, the application of the microfluidic proteomic reactor was demonstrated for the simultaneous processing of the interactome of the histone variant Htz1 in wild-type yeast and in a swr1Δ yeast strain compared to an untagged control using a novel three-channel microfluidic proteomic reactor. PMID:21520965

  19. X-ray transparent Microfluidics for Protein Crystallization and Biomineralization

    NASA Astrophysics Data System (ADS)

    Opathalage, Achini

    Protein crystallization demands the fundamental understanding of nucleation and applying techniques to find the optimal conditions to achieve the kinetic pathway for a large and defect free crystal. Classical nucleation theory predicts that the nucleation occurs at high supersaturation conditions. In this dissertation we sought out to develop techniques to attain optimal supersaturation profile to a large defect free crystal and subject it to in-situ X-ray diffraction using microfluidics. We have developed an emulsion-based serial crystallographic technology in nanolitre-sized droplets of protein solution encapsulated in to nucleate one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different un-oriented crystals. As proof of concept, the structure of Glucose Isomerase was solved to 2.1 A. We have developed a suite of X-ray semi-transparent micrfluidic devices which enables; controlled evaporation as a method of increasing supersaturation and manipulating the phase space of proteins and small molecules. We exploited the inherently high water permeability of the thin X-ray semi-transparent devices as a mean of increasing the supersaturation by controlling the evaporation. We fabricated the X-ray semi-transparent version of the PhaseChip with a thin PDMS membrane by which the storage and the reservoir layers are separated, and studies the phase transition of amorphous CaCO3.

  20. Low-cost, high-throughput fabrication of cloth-based microfluidic devices using a photolithographical patterning technique.

    PubMed

    Wu, Peijing; Zhang, Chunsun

    2015-03-21

    In this work, we first report a facile, low-cost and high-throughput method for photolithographical fabrication of microfluidic cloth-based analytical devices (μCADs) by simply using a cotton cloth as a substrate material and employing an inexpensive hydrophobic photoresist laboratory-formulated from commercially available reagents, which allows patterning of reproducible hydrophilic-hydrophobic features in the cloth with well-defined and uniform boundaries. Firstly, we evaluated the wicking properties of cotton cloths by testing the wicking rate in the cloth channel, in combination with scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analyses. It is demonstrated that the wicking properties of the cloth microfluidic channel can be improved by soaking the cloth substrate in 20 wt% NaOH solution and by washing the cloth-based microfluidic patterns with 3 wt% SDS solution. Next, we studied the minimum dimensions achievable for the width of the hydrophobic barriers and hydrophilic channels. The results indicate that the smallest width for a desired hydrophobic barrier is designed to be 100 μm and that for a desired hydrophilic channel is designed to be 500 μm. Finally, the high-throughput μCADs prepared using the developed fabrication technique were demonstrated for colorimetric assays of glucose and protein in artificial urine samples. It has been shown that the photolithographically patterned μCADs have potential for a simple, quantitative colorimetric urine test. The combination of cheap cloth and inexpensive high-throughput photolithography enables the development of new types of low-cost cloth-based microfluidic devices, such as "microzone plates" and "gate arrays", which provide new methods to perform biochemical assays or control fluid flow. PMID:25656508

  1. 3D-printed microfluidic chips with patterned, cell-laden hydrogel constructs.

    PubMed

    Knowlton, Stephanie; Yu, Chu Hsiang; Ersoy, Fulya; Emadi, Sharareh; Khademhosseini, Ali; Tasoglu, Savas

    2016-06-01

    Three-dimensional (3D) printing offers potential to fabricate high-throughput and low-cost fabrication of microfluidic devices as a promising alternative to traditional techniques which enables efficient design iterations in the development stage. In this study, we demonstrate a single-step fabrication of a 3D transparent microfluidic chip using two alternative techniques: a stereolithography-based desktop 3D printer and a two-step fabrication using an industrial 3D printer based on polyjet technology. This method, compared to conventional fabrication using relatively expensive materials and labor-intensive processes, presents a low-cost, rapid prototyping technique to print functional 3D microfluidic chips. We enhance the capabilities of 3D-printed microfluidic devices by coupling 3D cell encapsulation and spatial patterning within photocrosslinkable gelatin methacryloyl (GelMA). The platform presented here serves as a 3D culture environment for long-term cell culture and growth. Furthermore, we have demonstrated the ability to print complex 3D microfluidic channels to create predictable and controllable fluid flow regimes. Here, we demonstrate the novel use of 3D-printed microfluidic chips as controllable 3D cell culture environments, advancing the applicability of 3D printing to engineering physiological systems for future applications in bioengineering. PMID:27321481

  2. Flexible microfluidic cloth-based analytical devices using a low-cost wax patterning technique.

    PubMed

    Nilghaz, Azadeh; Wicaksono, Dedy H B; Gustiono, Dwi; Abdul Majid, Fadzilah Adibah; Supriyanto, Eko; Abdul Kadir, Mohammed Rafiq

    2012-01-01

    This paper describes the fabrication of microfluidic cloth-based analytical devices (μCADs) using a simple wax patterning method on cotton cloth for performing colorimetric bioassays. Commercial cotton cloth fabric is proposed as a new inexpensive, lightweight, and flexible platform for fabricating two- (2D) and three-dimensional (3D) microfluidic systems. We demonstrated that the wicking property of the cotton microfluidic channel can be improved by scouring in soda ash (Na(2)CO(3)) solution which will remove the natural surface wax and expose the underlying texture of the cellulose fiber. After this treatment, we fabricated narrow hydrophilic channels with hydrophobic barriers made from patterned wax to define the 2D microfluidic devices. The designed pattern is carved on wax-impregnated paper, and subsequently transferred to attached cotton cloth by heat treatment. To further obtain 3D microfluidic devices having multiple layers of pattern, a single layer of wax patterned cloth can be folded along a predefined folding line and subsequently pressed using mechanical force. All the fabrication steps are simple and low cost since no special equipment is required. Diagnostic application of cloth-based devices is shown by the development of simple devices that wick and distribute microvolumes of simulated body fluids along the hydrophilic channels into reaction zones to react with analytical reagents. Colorimetric detection of bovine serum albumin (BSA) in artificial urine is carried out by direct visual observation of bromophenol blue (BPB) colour change in the reaction zones. Finally, we show the flexibility of the novel microfluidic platform by conducting a similar reaction in a bent pinned μCAD. PMID:22089026

  3. Accessing Protein Methyltransferase and Demethylase Enzymology Using Microfluidic Capillary Electrophoresis

    PubMed Central

    Wigle, Tim J.; Provencher, Laurel M.; Norris, Jacqueline L.; Jin, Jian; Brown, Peter J.; Frye, Stephen V.; Janzen, William P.

    2010-01-01

    Summary The discovery of small molecules targeting the > 80 enzymes that add (methyltransferases) or remove (demethylases) methyl marks from lysine and arginine residues, most notably present in histone tails, may yield unprecedented chemotherapeutic agents and facilitate regenerative medicine. To better enable chemical exploration of these proteins, we have developed a novel and highly quantitative microfluidic capillary electrophoresis assay to enable full mechanistic studies of these enzymes and the kinetics of their inhibition. This technology separates small biomolecules, i.e., peptides, based on their charge-to-mass ratio. Methylation, however, does not alter the charge of peptide substrates. To overcome this limitation, we have employed a methylation-sensitive endoproteinase strategy to separate methylated from unmethylated peptides. The assay was validated on a lysine methyltransferase (G9a) and a lysine demethylase (LSD1) and was employed to investigate the inhibition of G9a by small molecules. PMID:20659682

  4. Patterned adhesive enables construction of nonplanar three-dimensional paper microfluidic circuits.

    PubMed

    Kalish, Brent; Tsutsui, Hideaki

    2014-11-21

    This article discusses the fabrication of planar and nonplanar 3D paper microfluidic circuits through the use of patterned spray adhesive application and origami techniques. The individual paper layers are held together via semi-permanent adhesive bonds without the need for external clamps. Semi-permanent bonds accommodate the repeated folding and unfolding required by complex origami device structures and allow the device to be unfolded post-use to view internally displayed results. Combinations of adhesive patterns and fluid channel widths were identified that did not prevent the fluid from traveling between layers and through the entire circuit. Further, this method was extended to nonplanar 3D paper microfluidic circuits, demonstrated via multi-fluid wicking within a modified origami peacock. Such nonplanar 3D paper microfluidic circuits are expected to offer an entirely new platform for exploring new designs and functions of paper analytical devices. PMID:25222567

  5. Microfluidic Western blotting of low-molecular-mass proteins.

    PubMed

    Gerver, Rachel E; Herr, Amy E

    2014-11-01

    We describe a microfluidic Western blot assay (μWestern) using a Tris tricine discontinuous buffer system suitable for analyses of a wide molecular mass range (6.5-116 kDa). The Tris tricine μWestern is completed in an enclosed, straight glass microfluidic channel housing a photopatterned polyacrylamide gel that incorporates a photoactive benzophenone methacrylamide monomer. Upon brief ultraviolet (UV) light exposure, the hydrogel toggles from molecular sieving for size-based separation to a covalent immobilization scaffold for in situ antibody probing. Electrophoresis controls all assay stages, affording purely electronic operation with no pumps or valves needed for fluid control. Electrophoretic introduction of antibody into and along the molecular sieving gel requires that the probe must traverse through (i) a discontinuous gel interface central to the transient isotachophoresis used to achieve high-performance separations and (ii) the full axial length of the separation gel. In-channel antibody probing of small molecular mass species is especially challenging, since the gel must effectively sieve small proteins while permitting effective probing with large-molecular-mass antibodies. To create a well-controlled gel interface, we introduce a fabrication method that relies on a hydrostatic pressure mismatch between the buffer and polymer precursor solution to eliminate the interfacial pore-size control issues that arise when a polymerizing polymer abuts a nonpolymerizing polymer solution. Combined with a new swept antibody probe plug delivery scheme, the Tris tricine μWestern blot enables 40% higher separation resolution as compared to a Tris glycine system, destacking of proteins down to 6.5 kDa, and a 100-fold better signal-to-noise ratio (SNR) for small pore gels, expanding the range of applicable biological targets. PMID:25268977

  6. Microfluidic Western Blotting of Low-Molecular-Mass Proteins

    PubMed Central

    2015-01-01

    We describe a microfluidic Western blot assay (μWestern) using a Tris tricine discontinuous buffer system suitable for analyses of a wide molecular mass range (6.5–116 kDa). The Tris tricine μWestern is completed in an enclosed, straight glass microfluidic channel housing a photopatterned polyacrylamide gel that incorporates a photoactive benzophenone methacrylamide monomer. Upon brief ultraviolet (UV) light exposure, the hydrogel toggles from molecular sieving for size-based separation to a covalent immobilization scaffold for in situ antibody probing. Electrophoresis controls all assay stages, affording purely electronic operation with no pumps or valves needed for fluid control. Electrophoretic introduction of antibody into and along the molecular sieving gel requires that the probe must traverse through (i) a discontinuous gel interface central to the transient isotachophoresis used to achieve high-performance separations and (ii) the full axial length of the separation gel. In-channel antibody probing of small molecular mass species is especially challenging, since the gel must effectively sieve small proteins while permitting effective probing with large-molecular-mass antibodies. To create a well-controlled gel interface, we introduce a fabrication method that relies on a hydrostatic pressure mismatch between the buffer and polymer precursor solution to eliminate the interfacial pore-size control issues that arise when a polymerizing polymer abuts a nonpolymerizing polymer solution. Combined with a new swept antibody probe plug delivery scheme, the Tris tricine μWestern blot enables 40% higher separation resolution as compared to a Tris glycine system, destacking of proteins down to 6.5 kDa, and a 100-fold better signal-to-noise ratio (SNR) for small pore gels, expanding the range of applicable biological targets. PMID:25268977

  7. Morphology-Patterned Anisotropic Wetting Surface for Fluid Control and Gas-Liquid Separation in Microfluidics.

    PubMed

    Wang, Shuli; Yu, Nianzuo; Wang, Tieqiang; Ge, Peng; Ye, Shunsheng; Xue, Peihong; Liu, Wendong; Shen, Huaizhong; Zhang, Junhu; Yang, Bai

    2016-05-25

    This article shows morphology-patterned stripes as a new platform for directing flow guidance of the fluid in microfluidic devices. Anisotropic (even unidirectional) spreading behavior due to anisotropic wetting of the underlying surface is observed after integrating morphology-patterned stripes with a Y-shaped microchannel. The anisotropic wetting flow of the fluid is influenced by the applied pressure, dimensions of the patterns, including the period and depth of the structure, and size of the channels. Fluids with different surface tensions show different flowing anisotropy in our microdevice. Moreover, the morphology-patterned surfaces could be used as a microvalve, and gas-water separation in the microchannel was realized using the unidirectional flow of water. Therefore, benefiting from their good performance and simple fabrication process, morphology-patterned surfaces are good candidates to be applied in controlling the fluid behavior in microfluidics. PMID:27128986

  8. Regioselective patterning of multiple SAMs and applications in surface-guided smart microfluidics.

    PubMed

    Chen, Chuanzhao; Xu, Pengcheng; Li, Xinxin

    2014-12-24

    A top-down nanofabrication technology is developed to integrate multiple SAMs (self-assembled monolayers) into regioselective patterns. With ultraviolet light exposure through regioselectively hollowed hard mask, an existing SAM at designated microregions can be removed and a dissimilar kind of SAM can be regrown there. By repeating the photolithography-like process cycle, diverse kinds of SAM building blocks can be laid out as a desired pattern in one microfluidic channel. In order to ensure high quality of the surface modifications, the SAMs are vapor-phase deposited before the channel is closed by a bonding process. For the first time the technique makes it possible to integrate three or more kinds of SAMs in one microchannel. The technique is very useful for multiplex surface functionalization of microfluidic chips where different segments of a microfluidic channel need to be individually modified with different SAMs or into arrayed pattern for surface-guided fluidic properties like hydrophobicity/philicity and/or oleophobicity/philicity, etc. The technique has been well validated by experimental demonstration of various surface-directed flow-guiding functions. By modifying a microchannel surface into an arrayed pattern of multi-SAM "two-tone" stripe array, surface-guiding-induced 3D swirling flow is generated in a microfluidic channel that experimentally exhibits quick oil/water mixing and high-efficiency oil-to-water chemical extraction. PMID:25438296

  9. A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins

    PubMed Central

    Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.

    2011-01-01

    Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells. PMID:21974603

  10. An optimized resistor pattern for temperature gradient control in microfluidics

    NASA Astrophysics Data System (ADS)

    Selva, Bertrand; Marchalot, Julien; Jullien, Marie-Caroline

    2009-06-01

    In this paper, we demonstrate the possibility of generating high-temperature gradients with a linear temperature profile when heating is provided in situ. Thanks to improved optimization algorithms, the shape of resistors, which constitute the heating source, is optimized by applying the genetic algorithm NSGA-II (acronym for the non-dominated sorting genetic algorithm) (Deb et al 2002 IEEE Trans. Evol. Comput. 6 2). Experimental validation of the linear temperature profile within the cavity is carried out using a thermally sensitive fluorophore, called Rhodamine B (Ross et al 2001 Anal. Chem. 73 4117-23, Erickson et al 2003 Lab Chip 3 141-9). The high level of agreement obtained between experimental and numerical results serves to validate the accuracy of this method for generating highly controlled temperature profiles. In the field of actuation, such a device is of potential interest since it allows for controlling bubbles or droplets moving by means of thermocapillary effects (Baroud et al 2007 Phys. Rev. E 75 046302). Digital microfluidics is a critical area in the field of microfluidics (Dreyfus et al 2003 Phys. Rev. Lett. 90 14) as well as in the so-called lab-on-a-chip technology. Through an example, the large application potential of such a technique is demonstrated, which entails handling a single bubble driven along a cavity using simple and tunable embedded resistors.

  11. A Microfluidic Platform for Characterization of Protein-Protein Interactions.

    PubMed

    Javanmard, Mehdi; Talasaz, Amirali H; Nemat-Gorgani, Mohsen; Huber, David E; Pease, Fabian; Ronaghi, Mostafa; Davis, Ronald W

    2009-08-01

    Traditionally, expensive and time consuming techniques such as mass spectrometry and Western Blotting have been used for characterization of protein-protein interactions. In this paper, we describe the design, fabrication, and testing of a rapid and inexpensive sensor, involving the use of microelectrodes in a microchannel, which can be used for real-time electrical detection of specific interactions between proteins. We have successfully demonstrated detection of target glycoprotein-glycoprotein interactions, antigen-antibody interactions, and glycoprotein-antigen interactions. We have also demonstrated the ability of this technique to distinguish between strong and weak interactions. Using this approach, it may be possible to multiplex an array of these sensors onto a chip and probe a complex mixture for various types of interactions involving protein molecules. PMID:20467571

  12. Multiplexed microfluidic blotting of proteins and nucleic acids by parallel, serpentine microchannels.

    PubMed

    He, Sha; Zhang, Yi; Wang, Pei; Xu, Xingzhi; Zhu, Kui; Pan, Wenying; Liu, Wenwen; Cai, Kaiyong; Sun, Jiashu; Zhang, Wei; Jiang, Xingyu

    2015-01-01

    This work develops a high-throughput, high-efficiency and straightforward microfluidic blotting method for analyzing proteins and nucleic acids. Sample solutions containing antibodies (for protein detection) or hybridization probes (for nucleic acid detection) are introduced into the parallel, serpentine microchannels to specifically recognize the immobilized targets on the substrate, achieving the identification of multiple targets in multiple samples simultaneously. The loading control, molecular weight markers, and antigen/antibody titration are designed and integrated into the microfluidic chip, thus allowing for the quantification of proteins and nucleic acids. Importantly, we could easily distinguish the adjacent blotting bands inside parallel microchannels, which may be difficult to achieve in conventional blotting. The small dimensions of microfluidic channels also help to reduce the amount of probing molecules and to accelerate the biochemical reaction. Our microfluidic blotting could bypass the steps of blocking and washing, further reducing the operation time and complexity. PMID:25342223

  13. A microfluidic, high throughput protein crystal growth method for microgravity.

    PubMed

    Carruthers, Carl W; Gerdts, Cory; Johnson, Michael D; Webb, Paul

    2013-01-01

    The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions' microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 10(3) cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories. PMID:24278480

  14. A Microfluidic, High Throughput Protein Crystal Growth Method for Microgravity

    PubMed Central

    Carruthers Jr, Carl W.; Gerdts, Cory; Johnson, Michael D.; Webb, Paul

    2013-01-01

    The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions’ microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 103 cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories. PMID:24278480

  15. Patterned fluoropolymer barriers for containment of organic solvents within paper-based microfluidic devices.

    PubMed

    Chen, Benny; Kwong, Philip; Gupta, Malancha

    2013-12-11

    In this study, we demonstrate for the first time the ability to pattern lipophobic fluoropolymer barriers for the incorporation of pure organic solvents as operating liquids within paper-based microfluidic devices. Our fabrication method involves replacing traditional wax barriers with fluoropolymer coatings by combining initiated chemical vapor deposition with inhibiting transition metal salt to pattern the polymer. Multiple techniques for patterning the transition metal salt are tested including painting, spray coating, and selective wetting through the use of a photoresist. The efficacy of the barrier coatings to contain organic solvents is found to be dependent on the conformality of the polymer deposited around the paper fibers. We demonstrate examples of the benefits provided by the containment of organic solvents in paper-based microfluidic applications including the ability to tune the separation of analytes by varying the operating solvent and by modifying the channel region of the devices with additional polymer coatings. The work exhibited in this paper has the potential to significantly expand the applications of paper-based microfluidics to include detection of water insoluble analytes. Additionally, the generality of the patterning process allows this technique to be extended to other applications that may require the use of patterned hydrophobic and lipophobic regions, such as biosensing, chemical detection, and optics. PMID:24283374

  16. Thermoplastic microfluidic devices and their applications in protein and DNA analysis

    PubMed Central

    Liu, Ke; Fan, Z. Hugh

    2013-01-01

    Microfluidics is a platform technology that has been used for genomics, proteomics, chemical synthesis, environment monitoring, cellular studies, and other applications. The fabrication materials of microfluidic devices have traditionally included silicon and glass, but plastics have gained increasing attention in the past few years. We focus this review on thermoplastic microfluidic devices and their applications in protein and DNA analysis. We outline the device design and fabrication methods, followed by discussion on the strategies of surface treatment. We then concentrate on several significant advancements in applying thermoplastic microfluidic devices to protein separation, immunoassays, and DNA analysis. Comparison among numerous efforts, as well as the discussion on the challenges and innovation associated with detection, is presented. PMID:21274478

  17. Tape Transfer Atomization Patterning of Liquid Alloys for Microfluidic Stretchable Wireless Power Transfer

    PubMed Central

    Jeong, Seung Hee; Hjort, Klas; Wu, Zhigang

    2015-01-01

    Stretchable electronics offers unsurpassed mechanical compliance on complex or soft surfaces like the human skin and organs. To fully exploit this great advantage, an autonomous system with a self-powered energy source has been sought for. Here, we present a new technology to pattern liquid alloys on soft substrates, targeting at fabrication of a hybrid-integrated power source in microfluidic stretchable electronics. By atomized spraying of a liquid alloy onto a soft surface with a tape transferred adhesive mask, a universal fabrication process is provided for high quality patterns of liquid conductors in a meter scale. With the developed multilayer fabrication technique, a microfluidic stretchable wireless power transfer device with an integrated LED was demonstrated, which could survive cycling between 0% and 25% strain over 1,000 times. PMID:25673261

  18. Tape transfer atomization patterning of liquid alloys for microfluidic stretchable wireless power transfer.

    PubMed

    Jeong, Seung Hee; Hjort, Klas; Wu, Zhigang

    2015-01-01

    Stretchable electronics offers unsurpassed mechanical compliance on complex or soft surfaces like the human skin and organs. To fully exploit this great advantage, an autonomous system with a self-powered energy source has been sought for. Here, we present a new technology to pattern liquid alloys on soft substrates, targeting at fabrication of a hybrid-integrated power source in microfluidic stretchable electronics. By atomized spraying of a liquid alloy onto a soft surface with a tape transferred adhesive mask, a universal fabrication process is provided for high quality patterns of liquid conductors in a meter scale. With the developed multilayer fabrication technique, a microfluidic stretchable wireless power transfer device with an integrated LED was demonstrated, which could survive cycling between 0% and 25% strain over 1,000 times. PMID:25673261

  19. Tape Transfer Atomization Patterning of Liquid Alloys for Microfluidic Stretchable Wireless Power Transfer

    NASA Astrophysics Data System (ADS)

    Jeong, Seung Hee; Hjort, Klas; Wu, Zhigang

    2015-02-01

    Stretchable electronics offers unsurpassed mechanical compliance on complex or soft surfaces like the human skin and organs. To fully exploit this great advantage, an autonomous system with a self-powered energy source has been sought for. Here, we present a new technology to pattern liquid alloys on soft substrates, targeting at fabrication of a hybrid-integrated power source in microfluidic stretchable electronics. By atomized spraying of a liquid alloy onto a soft surface with a tape transferred adhesive mask, a universal fabrication process is provided for high quality patterns of liquid conductors in a meter scale. With the developed multilayer fabrication technique, a microfluidic stretchable wireless power transfer device with an integrated LED was demonstrated, which could survive cycling between 0% and 25% strain over 1,000 times.

  20. Protein expression patterns of the yeast mating response.

    PubMed

    Yuan, Haiyu; Zhang, Rongfei; Shao, Bin; Wang, Xuan; Ouyang, Qi; Hao, Nan; Luo, Chunxiong

    2016-06-13

    Microfluidics, in combination with time-lapse microscopy, is a transformative technology that significantly enhances our ability to monitor and probe biological processes in living cells. However, high-throughput microfluidic devices mostly require sophisticated preparatory and setup work and are thus hard to adopt by non-experts. In this work, we designed an easy-to-use microfluidic chip, which enables tracking of 48 GFP-tagged yeast strains, with each strain under two different stimulus conditions, in a single experiment. We used this technology to investigate the dynamic pattern of protein expression during the yeast mating differentiation response. High doses of pheromone induce cell cycle arrest and the shmoo morphology, whereas low doses of pheromone lead to elongation and chemotrophic growth. By systematically analyzing the protein dynamics of 156 pheromone-regulated genes, we identified groups of genes that are preferentially induced in response to low-dose pheromone (elongation during growth) or high-dose pheromone (shmoo formation and cell cycle arrest). The protein dynamics of these genes may provide insights into the mechanisms underlying the differentiation switch induced by different doses of pheromone. PMID:27177258

  1. Topographically-patterned porous membranes in a microfluidic device as an in vitro model of renal reabsorptive barriers

    PubMed Central

    Frohlich, Else M.; Alonso, José Luis; Borenstein, Jeffrey T.; Zhang, Xin; Arnaout, M. Amin

    2015-01-01

    Models of reabsorptive barriers require both a means to provide realistic physiologic cues to and quantify transport across a layer of cells forming the barrier. Here we have topographically-patterned porous membranes with several user-defined pattern types. To demonstrate the utility of the patterned membranes, we selected one type of pattern and applied it to a membrane to serve as a cell culture support in a microfluidic model of a renal reabsorptive barrier. The topographic cues in the model resemble physiological cues found in vivo while the porous structure allows quantification of transport across the cell layer. Sub-micron surface topography generated via hot-embossing onto a track-etched polycarbonate membrane, fully replicated topographical features and preserved porous architecture. Pore size and shape were analyzed with SEM and image analysis to determine the effect of hot embossing on pore morphology. The membrane was assembled into a bilayer microfluidic device and a human kidney proximal tubule epithelial cell line (HK-2) and primary renal proximal tubule epithelial cells (RPTEC) were cultured to confluency on the membrane. Immunofluorescent staining of both cell types revealed protein expression indicative of the formation of a reabsorptive barrier responsive to mechanical stimulation: ZO-1 (tight junction), paxillin (focal adhesions) and acetylated α-tubulin (primary cilia). HK-2 and RPTEC aligned in the direction of ridge/groove topography of the membrane in the device, evidence that the device has mechanical control over cell response. This topographically-patterned porous membrane provides an in vitro platform on which to model reabsorptive barriers with meaningful applications for understanding biological transport phenomenon, underlying disease mechanisms, and drug toxicity. PMID:23636129

  2. 3-dimensional electrode patterning within a microfluidic channel using metal ion implantation.

    PubMed

    Choi, Jae-Woo; Rosset, Samuel; Niklaus, Muhamed; Adleman, James R; Shea, Herbert; Psaltis, Demetri

    2010-03-21

    The application of electrical fields within a microfluidic channel enables many forms of manipulation necessary for lab-on-a-chip devices. Patterning electrodes inside the microfluidic channel generally requires multi-step optical lithography. Here, we utilize an ion-implantation process to pattern 3D electrodes within a fluidic channel made of polydimethylsiloxane (PDMS). Electrode structuring within the channel is achieved by ion implantation at a 40 degrees angle with a metal shadow mask. The advantages of three-dimensional structuring of electrodes within a fluidic channel over traditional planar electrode designs are discussed. Two possible applications are presented: asymmetric particles can be aligned in any of the three axial dimensions with electro-orientation; colloidal focusing and concentration within a fluidic channel can be achieved through dielectrophoresis. Demonstrations are shown with E. coli, a rod shaped bacteria, and indicate the potential that ion-implanted microfluidic channels have for manipulations in the context of lab-on-a-chip devices. PMID:20221568

  3. Study of Spatio-Temporal Immunofluorescence on Bead Patterns in a Microfluidic Channel

    NASA Astrophysics Data System (ADS)

    Sivagnanam, Venkataragavalu; Yang, Hui; Gijs, Martin A. M.

    2010-12-01

    We performed a direct immunoassay inside a microfluidic channel on patterned streptavidin-coated beads, which captured fluorescently-labeled biotin target molecules from a continuous flow. We arranged the beads in a dot array at the bottom of the channel and demonstrated their position- and flow rate-dependent fluorescence. As the target analyte gets gradually depleted from the flow when passing downstream the channel, the highest fluorescence intensity was observed on the most upstream positioned dot patterns. We propose a simple analytical convection model to explain this spatio-temporal fluorescence.

  4. An integrated microfluidic biochemical detection system for protein analysis with magnetic bead-based sampling capabilities.

    PubMed

    Choi, Jin-Woo; Oh, Kwang W; Thomas, Jennifer H; Heineman, William R; Halsall, H Brian; Nevin, Joseph H; Helmicki, Arthur J; Henderson, H Thurman; Ahn, Chong H

    2002-02-01

    This paper presents the development and characterization of an integrated microfluidic biochemical detection system for fast and low-volume immunoassays using magnetic beads, which are used as both immobilization surfaces and bio-molecule carriers. Microfluidic components have been developed and integrated to construct a microfluidic biochemical detection system. Magnetic bead-based immunoassay, as a typical example of biochemical detection and analysis, has been successfully performed on the integrated microfluidic biochemical analysis system that includes a surface-mounted biofilter and electrochemical sensor on a glass microfluidic motherboard. Total time required for an immunoassay was less than 20 min including sample incubation time, and sample volume wasted was less than 50 microl during five repeated assays. Fast and low-volume biochemical analysis has been successfully achieved with the developed biofilter and immunosensor, which is integrated to the microfluidic system. Such a magnetic bead-based biochemical detection system, described in this paper, can be applied to protein analysis systems. PMID:15100857

  5. Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array

    SciTech Connect

    Lyubimov, Artem Y.; Murray, Thomas D.; Koehl, Antoine; Araci, Ismail Emre; Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Cohen, Aina E.; Soltis, S. Michael; Baxter, Elizabeth L.; Brewster, Aaron S.; Sauter, Nicholas K.; Brunger, Axel T.; Berger, James M.

    2015-04-01

    A microfluidic platform has been developed for the capture and X-ray analysis of protein microcrystals, affording a means to improve the efficiency of XFEL and synchrotron experiments. X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.

  6. Understanding wax screen-printing: a novel patterning process for microfluidic cloth-based analytical devices.

    PubMed

    Liu, Min; Zhang, Chunsun; Liu, Feifei

    2015-09-01

    In this work, we first introduce the fabrication of microfluidic cloth-based analytical devices (μCADs) using a wax screen-printing approach that is suitable for simple, inexpensive, rapid, low-energy-consumption and high-throughput preparation of cloth-based analytical devices. We have carried out a detailed study on the wax screen-printing of μCADs and have obtained some interesting results. Firstly, an analytical model is established for the spreading of molten wax in cloth. Secondly, a new wax screen-printing process has been proposed for fabricating μCADs, where the melting of wax into the cloth is much faster (∼5 s) and the heating temperature is much lower (75 °C). Thirdly, the experimental results show that the patterning effects of the proposed wax screen-printing method depend to a certain extent on types of screens, wax melting temperatures and melting time. Under optimized conditions, the minimum printing width of hydrophobic wax barrier and hydrophilic channel is 100 μm and 1.9 mm, respectively. Importantly, the developed analytical model is also well validated by these experiments. Fourthly, the μCADs fabricated by the presented wax screen-printing method are used to perform a proof-of-concept assay of glucose or protein in artificial urine with rapid high-throughput detection taking place on a 48-chamber cloth-based device and being performed by a visual readout. Overall, the developed cloth-based wax screen-printing and arrayed μCADs should provide a new research direction in the development of advanced sensor arrays for detection of a series of analytes relevant to many diverse applications. PMID:26388382

  7. Microfluidics for the analysis of membrane proteins: how do we get there?

    PubMed

    Battle, Katrina N; Uba, Franklin I; Soper, Steven A

    2014-08-01

    The development of fully automated and high-throughput systems for proteomics is now in demand because of the need to generate new protein-based disease biomarkers. Unfortunately, it is difficult to identify protein biomarkers that are low abundant when in the presence of highly abundant proteins, especially in complex biological samples such as serum, cell lysates, and other biological fluids. Membrane proteins, which are in many cases of low abundance compared to the cytosolic proteins, have various functions and can provide insight into the state of a disease and serve as targets for new drugs making them attractive biomarker candidates. Traditionally, proteins are identified through the use of gel electrophoretic techniques, which are not always suitable for particular protein samples such as membrane proteins. Microfluidics offers the potential as a fully automated platform for the efficient and high-throughput analysis of complex samples, such as membrane proteins, and do so with performance metrics that exceed their bench-top counterparts. In recent years, there have been various improvements to microfluidics and their use for proteomic analysis as reported in the literature. Consequently, this review presents an overview of the traditional proteomic-processing pipelines for membrane proteins and insights into new technological developments with a focus on the applicability of microfluidics for the analysis of membrane proteins. Sample preparation techniques will be discussed in detail and novel interfacing strategies as it relates to MS will be highlighted. Lastly, some general conclusions and future perspectives are presented. PMID:24585436

  8. Fabrication of thermo-responsive microfluidic membrane using photopolymerization patterning

    NASA Astrophysics Data System (ADS)

    Kim, Hyejeong; Lee, Sang Joon

    2015-11-01

    The programmed manipulation of responsive functional hydrogels is receiving large attention because of its unique functions and wide range of engineering applications. In this study, we developed an innovative stomata-inspired membrane (SIM) by fabricating a temperature-responsive hydrogel with a simple, cost effective, and high-throughput photopolymerization patterning process. Polymerization-induced diffusion on the macro-scale surface gives rise to form a multi-parted polymer membrane with fine pores by simple UV irradiation. After heating the SIM, the less deformable thick frame supports the whole structure, and the highly deformable thin base regulates the size of pores. The morphological configuration of the SIM can be easily changed by varying the solution composition or selecting a suitable photomask with different pattern. The developed SIM has the special sensing-to-actuation functions of stimuli-responsive hydrogels. This membrane with temperature-responsive pores would be potentially utilized in numerous practical applications, such as filter membranes with self-adjustable pores, membrane-based sensors, membrane-based actuators, and multi-functional membranes etc. This study was supported by the National Research Foundation of Korea (NRF) and funded by the Korean government (MSIP) (Grant No. 2008-0061991).

  9. Paramagnetic Structures within a Microfluidic Channel for Enhanced Immunomagnetic Isolation and Surface Patterning of Cells

    PubMed Central

    Sun, Chen; Hassanisaber, Hamid; Yu, Richard; Ma, Sai; Verbridge, Scott S.; Lu, Chang

    2016-01-01

    In this report, we demonstrate a unique method for embedding magnetic structures inside a microfluidic channel for cell isolation. We used a molding process to fabricate these structures out of a ferrofluid of cobalt ferrite nanoparticles. We show that the embedded magnetic structures significantly increased the magnetic field in the channel, resulting in up to 4-fold enhancement in immunomagnetic capture as compared with a channel without these embedded magnetic structures. We also studied the spatial distribution of trapped cells both experimentally and computationally. We determined that the surface pattern of these trapped cells was determined by both location of the magnet and layout of the in-channel magnetic structures. Our magnetic structure embedded microfluidic device achieved over 90% capture efficiency at a flow velocity of 4 mm/s, a speed that was roughly two orders of magnitude faster than previous microfluidic systems used for a similar purpose. We envision that our technology will provide a powerful tool for detection and enrichment of rare cells from biological samples. PMID:27388549

  10. TiO2 coated microfluidic devices for recoverable hydrophilic and hydrophobic patterns

    NASA Astrophysics Data System (ADS)

    Lee, Jin-Hyung; Kim, Sang Kyung; Park, Hyung-Ho; Kim, Tae Song

    2015-03-01

    We report a simple method for modifying the surfaces of plastic microfluidic devices through dynamic coating process with a nano-colloidal TiO2 sol. The surface of the thermoplastic, cyclic olefin copolymer (COC) was coated with the TiO2 film, that displayed an effective photocatalytic property. The hydrophilic surface is obtained in the TiO2-coated zone of a microfluidic channel, and TiO2 coated surface degradation can be reversed easily by UV irradiation. The present work shows a photocatalytic activity concerning the effect of TiO2 coating density, which is controlled by the number of coating cycles. The hydrophilized surface was characterized by the contact angle of water and the TiO2 coated COC surface reduced the water contact angle from 85° to less than 10° upon UV irradiation. The photocatalytic effect of the layer that was coated five times with TiO2 was excellent, and the super-hydrophilicity of the TiO2 surface could be promptly recovered after 10 months of storage at atmospheric conditions. The COC microfluidic devices, in which TiO2 has been freshly deposited and aged for 10 months, were capable of generating water-in oil-in water (W/O/W) double emulsions easily and uniformly by simple control of the flow rates for demonstration of excellent hydrophilic patterning and recovery of the TiO2 coated in the microchannels.

  11. Paramagnetic Structures within a Microfluidic Channel for Enhanced Immunomagnetic Isolation and Surface Patterning of Cells

    NASA Astrophysics Data System (ADS)

    Sun, Chen; Hassanisaber, Hamid; Yu, Richard; Ma, Sai; Verbridge, Scott S.; Lu, Chang

    2016-07-01

    In this report, we demonstrate a unique method for embedding magnetic structures inside a microfluidic channel for cell isolation. We used a molding process to fabricate these structures out of a ferrofluid of cobalt ferrite nanoparticles. We show that the embedded magnetic structures significantly increased the magnetic field in the channel, resulting in up to 4-fold enhancement in immunomagnetic capture as compared with a channel without these embedded magnetic structures. We also studied the spatial distribution of trapped cells both experimentally and computationally. We determined that the surface pattern of these trapped cells was determined by both location of the magnet and layout of the in-channel magnetic structures. Our magnetic structure embedded microfluidic device achieved over 90% capture efficiency at a flow velocity of 4 mm/s, a speed that was roughly two orders of magnitude faster than previous microfluidic systems used for a similar purpose. We envision that our technology will provide a powerful tool for detection and enrichment of rare cells from biological samples.

  12. Paramagnetic Structures within a Microfluidic Channel for Enhanced Immunomagnetic Isolation and Surface Patterning of Cells.

    PubMed

    Sun, Chen; Hassanisaber, Hamid; Yu, Richard; Ma, Sai; Verbridge, Scott S; Lu, Chang

    2016-01-01

    In this report, we demonstrate a unique method for embedding magnetic structures inside a microfluidic channel for cell isolation. We used a molding process to fabricate these structures out of a ferrofluid of cobalt ferrite nanoparticles. We show that the embedded magnetic structures significantly increased the magnetic field in the channel, resulting in up to 4-fold enhancement in immunomagnetic capture as compared with a channel without these embedded magnetic structures. We also studied the spatial distribution of trapped cells both experimentally and computationally. We determined that the surface pattern of these trapped cells was determined by both location of the magnet and layout of the in-channel magnetic structures. Our magnetic structure embedded microfluidic device achieved over 90% capture efficiency at a flow velocity of 4 mm/s, a speed that was roughly two orders of magnitude faster than previous microfluidic systems used for a similar purpose. We envision that our technology will provide a powerful tool for detection and enrichment of rare cells from biological samples. PMID:27388549

  13. MICROFLUIDIC MIXERS FOR THE INVESTIGATION OF PROTEIN FOLDING USING SYNCHROTRON RADIATION CIRCULAR DICHROISM SPECTROSCOPY

    SciTech Connect

    Kane, A; Hertzog, D; Baumgartel, P; Lengefeld, J; Horsley, D; Schuler, B; Bakajin, O

    2006-03-20

    The purpose of this study is to design, fabricate and optimize microfluidic mixers to investigate the kinetics of protein secondary structure formation with Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. The mixers are designed to rapidly initiate protein folding reaction through the dilution of denaturant. The devices are fabricated out of fused silica, so that they are transparent in the UV. We present characterization of mixing in the fabricated devices, as well as the initial SRCD data on proteins inside the mixers.

  14. A Microfluidic Platform for High-Throughput Multiplexed Protein Quantitation

    PubMed Central

    Volpetti, Francesca; Garcia-Cordero, Jose; Maerkl, Sebastian J.

    2015-01-01

    We present a high-throughput microfluidic platform capable of quantitating up to 384 biomarkers in 4 distinct samples by immunoassay. The microfluidic device contains 384 unit cells, which can be individually programmed with pairs of capture and detection antibody. Samples are quantitated in each unit cell by four independent MITOMI detection areas, allowing four samples to be analyzed in parallel for a total of 1,536 assays per device. We show that the device can be pre-assembled and stored for weeks at elevated temperature and we performed proof-of-concept experiments simultaneously quantitating IL-6, IL-1β, TNF-α, PSA, and GFP. Finally, we show that the platform can be used to identify functional antibody combinations by screening 64 antibody combinations requiring up to 384 unique assays per device. PMID:25680117

  15. Optically Induced Thermal Gradients for Protein Characterization in Nanolitre-scale Samples in Microfluidic Devices

    PubMed Central

    Sagar, D. M.; Aoudjane, Samir; Gaudet, Matthieu; Aeppli, Gabriel; Dalby, Paul A.

    2013-01-01

    Proteins are the most vital biological functional units in every living cell. Measurement of protein stability is central to understanding their structure, function and role in diseases. While proteins are also sought as therapeutic agents, they can cause diseases by misfolding and aggregation in vivo. Here we demonstrate a novel method to measure protein stability and denaturation kinetics, on unprecedented timescales, through optically-induced heating of nanolitre samples in microfluidic capillaries. We obtain protein denaturation kinetics as a function of temperature, and accurate thermodynamic stability data, from a snapshot experiment on a single sample. We also report the first experimental characterization of optical heating in controlled microcapillary flow, verified by computational fluid dynamics modelling. Our results demonstrate that we now have the engineering science in hand to design integrated all-optical microfluidic chips for a diverse range of applications including in-vitro DNA amplification, healthcare diagnostics, and flow chemistry. PMID:23823279

  16. A perfusion-capable microfluidic bioreactor for assessing microbial heterologous protein production.

    PubMed

    Mozdzierz, Nicholas J; Love, Kerry R; Lee, Kevin S; Lee, Harry L T; Shah, Kartik A; Ram, Rajeev J; Love, J Christopher

    2015-07-21

    We present an integrated microfluidic bioreactor for fully continuous perfusion cultivation of suspended microbial cell cultures. This system allowed continuous and stable heterologous protein expression by sustaining the cultivation of Pichia pastoris over 11 days. This technical capability also allowed testing the impact of perfusion conditions on protein expression. This advance should enable small-scale models for process optimization in continuous biomanufacturing. PMID:26055071

  17. Cyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffraction

    SciTech Connect

    Emamzadah, Soheila; Petty, Tom J.; De Almeida, Victor; Nishimura, Taisuke; Joly, Jacques; Ferrer, Jean-Luc; Halazonetis, Thanos D.

    2009-09-01

    A cyclic olefin homopolymer-based microfluidics system has been established for protein crystallization and in situ X-ray diffraction. Microfluidics is a promising technology for the rapid identification of protein crystallization conditions. However, most of the existing systems utilize silicone elastomers as the chip material which, despite its many benefits, is highly permeable to water vapour. This limits the time available for protein crystallization to less than a week. Here, the use of a cyclic olefin homopolymer-based microfluidics system for protein crystallization and in situ X-ray diffraction is described. Liquid handling in this system is performed in 2 mm thin transparent cards which contain 500 chambers, each with a volume of 320 nl. Microbatch, vapour-diffusion and free-interface diffusion protocols for protein crystallization were implemented and crystals were obtained of a number of proteins, including chicken lysozyme, bovine trypsin, a human p53 protein containing both the DNA-binding and oligomerization domains bound to DNA and a functionally important domain of Arabidopsis Morpheus’ molecule 1 (MOM1). The latter two polypeptides have not been crystallized previously. For X-ray diffraction analysis, either the cards were opened to allow mounting of the crystals on loops or the crystals were exposed to X-rays in situ. For lysozyme, an entire X-ray diffraction data set at 1.5 Å resolution was collected without removing the crystal from the card. Thus, cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts.

  18. A perfusion-capable microfluidic bioreactor for assessing microbial heterologous protein production

    PubMed Central

    Mozdzierz, Nicholas J.; Love, Kerry R.; Lee, Kevin S.; Lee, Harry L. T.; Shah, Kartik A.; Ram, Rajeev J.

    2015-01-01

    We present an integrated microfluidic bioreactor for fully continuous perfusion cultivation of suspended microbial cell cultures. This system allowed continuous and stable heterologous protein expression by sustaining the cultivation of Pichia pastoris over 11 days. This technical capability also allowed testing the impact of perfusion conditions on protein expression. This advance should enable small-scale models for process optimization in continuous biomanufacturing. PMID:26055071

  19. Dynamics of Drosophila embryonic patterning network perturbed in space and time using microfluidics

    PubMed Central

    Lucchetta, Elena M.; Lee, Ji Hwan; Fu, Lydia A.; Patel, Nipam H.; Ismagilov, Rustem F.

    2009-01-01

    Biochemical networks are perturbed both by fluctuations in environmental conditions and genetic variation. These perturbations must be compensated for, especially when they occur during embryonic pattern formation. Complex chemical reaction networks displaying spatiotemporal dynamics have been controlled and understood by perturbing their environment in space and time1–3. Here, we apply this approach using microfluidics to investigate the robust network in Drosophila melanogaster that compensates for variation in the Bicoid morphogen gradient. We show that the compensation system can counteract the effects of extremely unnatural environmental conditions—a temperature step—in which the anterior and posterior halves of the embryo are developing at different temperatures and thus at different rates. Embryonic patterning was normal under this condition, suggesting that a simple reciprocal gradient system is not the mechanism of compensation. Time-specific reversals of the temperature step narrowed down the critical period for compensation to between 65 and 100 min after onset of embryonic development. The microfluidic technology used here may prove useful to future studies, as it allows spatial and temporal regulation of embryonic development. PMID:15858575

  20. Fabrication of microfluidic chips using lithographic patterning and adhesive bonding of the thick negative photoresist AZ 125 nXT

    NASA Astrophysics Data System (ADS)

    Knoll, Thorsten; Bergmann, Andreas; Nußbaum, Dominic

    2015-05-01

    In this work, for the first time the negative photoresist AZ 125 nXT was used for the fabrication of a microfluidic chip. Usually, fabrication of microfluidic devices on the basis of silicon or glass substrates is done by using the epoxy-based negative photoresist SU-8 or other thick film polymer materials. The suitability of SU-8 for various microfluidic applications has been shown in the fields of bioanalytic devices, lab-on-chip systems or microreaction technology. However, processing is always a very challenging task with regard to the adaptation of process parameters to the individual design and required functionality. Now, the AZ 125 nXT allows for the fabrication of structures in a wide thickness range with only one type of viscosity. In contrast to SU-8, the AZ 125 nXT is fully cross-linked during UV exposure and does not require a time-consuming post-exposure bake. 90 μm deep microfluidic channels were defined by lithographic patterning of AZ 125 nXT. Sealing of the open microfluidic channels was performed by a manual adhesive bonding process at a temperature of 100 °C. The fluidic function was successfully tested with flow rates up to 20 ml/min by means of a microfluidic edge connector. Long term stability and chemical resistance of the fabricated microfluidic channels will be investigated in the near future. The presented work shows the potential of AZ 125 nXT as a possible alternative to SU-8 for the fabrication of microfluidic chips.

  1. Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array

    DOE PAGESBeta

    Lyubimov, Artem Y.; Murray, Thomas D.; Koehl, Antoine; Araci, Ismail Emre; Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Cohen, Aina E.; Soltis, S. Michael; Baxter, Elizabeth L.; Brewster, Aaron S.; et al

    2015-03-27

    X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat formore » conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.« less

  2. Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array.

    PubMed

    Lyubimov, Artem Y; Murray, Thomas D; Koehl, Antoine; Araci, Ismail Emre; Uervirojnangkoorn, Monarin; Zeldin, Oliver B; Cohen, Aina E; Soltis, S Michael; Baxter, Elizabeth L; Brewster, Aaron S; Sauter, Nicholas K; Brunger, Axel T; Berger, James M

    2015-04-01

    X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources. PMID:25849403

  3. Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array

    SciTech Connect

    Lyubimov, Artem Y.; Murray, Thomas D.; Koehl, Antoine; Araci, Ismail Emre; Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Cohen, Aina E.; Soltis, S. Michael; Baxter, Elizabeth L.; Brewster, Aaron S.; Sauter, Nicholas K.; Brunger, Axel T.; Berger, James M.

    2015-03-27

    X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.

  4. Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array

    PubMed Central

    Lyubimov, Artem Y.; Murray, Thomas D.; Koehl, Antoine; Araci, Ismail Emre; Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Cohen, Aina E.; Soltis, S. Michael; Baxter, Elizabeth L.; Brewster, Aaron S.; Sauter, Nicholas K.; Brunger, Axel T.; Berger, James M.

    2015-01-01

    X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources. PMID:25849403

  5. A microfluidic linear node array for the study of protein-ligand interactions.

    PubMed

    Li, Cheuk-Wing; Yu, Guodong; Jiang, Jingyun; Lee, Simon Ming-Yuen; Yi, Changqing; Yue, Wanqing; Yang, Mengsu

    2014-10-21

    We have developed a microfluidic device for the continuous separation of small molecules from a protein mixture and demonstrated its practical use in the study of protein-ligand binding, a crucial aspect in drug discovery. Our results demonstrated dose-dependent binding between bovine serum albumin (BSA) and its small-molecule site marker, Eosin Y (EY), and found that the binding reached a plateau when the BSA : EY ratio was above 1, which agreed with the eosin binding capacity of BSA reported in literature. By streamline control using a combination of two fundamental building blocks (R and L nodes) with a microdevice operated at a high flow rate (up to 1300 μL h(-1)), a solution barrier was created to "filter" off protein/protein-ligand complexes such that the small unbound molecules were isolated and quantified easily. The percentage decrease of small molecules with increasing protein concentration indicated the presence of binding events. Several fluorophores with different molecular weights were used to test the performance of the microfluidic "filter", which was tunable by 1) the total flow rate, and/or 2) the flow distribution ratio between the two device inlets; both were easily controllable by changing the syringe pump settings. Since the microdevice was operated at a relatively high flow rate, aliquots were easily recovered from the device outlets to facilitate off-chip detection. This microfluidic design is a novel and promising tool for preliminary drug screening. PMID:25140880

  6. Fluorescence enhancement and multiple protein detection in ZnO nanostructure microfluidic devices.

    PubMed

    Sang, Chen-Hsiang; Chou, Shu-Jen; Pan, F M; Sheu, Jeng-Tzong

    2016-01-15

    In this study, different morphological ZnO nanostructures, those of sharp nanowires (NWs), rod NWs, and hexahedral-puncheon nanostructures, were grown in microfluidic channels on the same glass substrate. Characterizations of correspondent biomolecule binding properties were simulated and demonstrated. The surface was modified using 3-ammineopropyl-triethoxysilane (3-APTES) and biotin-N-hydroxysuccinimide ester (NHS-biotin). Different concentrations (4.17pM to 41.7nM) of dye-conjugated streptavidin were simultaneously infused through the second microfluidic channels, which lie 90° from the first microfluidic channels. The florescent intensity at the crossover areas showed good agreement with simulations, with sharp ZnO NWs exhibiting the largest dynamic range and the highest fluorescent intensity. We further characterize correspondent protein detection using sharp ZnO NWs. The surfaces of these ZnO NWs were modified with mouse immunoglobulin G (IgG), infused through the second microfluidic channels with dye-conjugated (Alexa 546) anti-mouse IgG in different concentrations. Concentrations ranging from 417fM to 41.7nM can be resolved using sharp ZnO NWs. Finally, multiple protein detection was demonstrated using a five-by-eight microfluidic channel array. Fluorescence images present clear multiple detections at the crossover areas when using the sharp ZnO NWs for simultaneous dye-conjugated anti-mouse IgG and dye-conjugated anti-rabbit IgG (Alexa 647) detection. PMID:26322591

  7. Recombinant Protein-Stabilized Monodisperse Microbubbles with Tunable Size Using a Valve-Based Microfluidic Device

    PubMed Central

    2015-01-01

    Microbubbles are used as contrast enhancing agents in ultrasound sonography and more recently have shown great potential as theranostic agents that enable both diagnostics and therapy. Conventional production methods lead to highly polydisperse microbubbles, which compromise the effectiveness of ultrasound imaging and therapy. Stabilizing microbubbles with surfactant molecules that can impart functionality and properties that are desirable for specific applications would enhance the utility of microbubbles. Here we generate monodisperse microbubbles with a large potential for functionalization by combining a microfluidic method and recombinant protein technology. Our microfluidic device uses an air-actuated membrane valve that enables production of monodisperse microbubbles with narrow size distribution. The size of microbubbles can be precisely tuned by dynamically changing the dimension of the channel using the valve. The microbubbles are stabilized by an amphiphilic protein, oleosin, which provides versatility in controlling the functionalization of microbubbles through recombinant biotechnology. We show that it is critical to control the composition of the stabilizing agents to enable formation of highly stable and monodisperse microbubbles that are echogenic under ultrasound insonation. Our protein-shelled microbubbles based on the combination of microfluidic generation and recombinant protein technology provide a promising platform for ultrasound-related applications. PMID:25265041

  8. Maximizing Fibroblast Adhesion on Protein-Coated Surfaces Using Microfluidic Cell Printing

    PubMed Central

    Davidoff, S.N.; Au, D.; Gale, B.K.; Brooks, B.D.; Brooks, A.E.

    2015-01-01

    translation of in vitro cell based assays to in vivo cellular response is imprecise at best. The advent of three-dimensional cell cultures in addition to bioreactor type microfluidics has improved the situation. However, these technical advances cannot be easily combined due to practical limitations. Development of a vertical microfluidic cell printer overcomes this obstacle, providing the ability to more closely recapitulate complex cellular environments and responses. As a proof of concept, we investigated the adhesion of fibroblasts under flow on protein-coated surfaces using a novel vertical microfluidic print head to isolate and manipulate both mechanical and biological factors as a model of fibroblast behavior during the foreign body response following implant insertion. A low flow rate with larger microfluidic channels onto a serum-coated surface has been determined to allow the highest density of viable fibroblasts to attach to the surface. While these insights into fibroblast surface attachment may lead to better material designs, the methods developed herein will certainly be useful as a biomaterials testing platform. PMID:26989480

  9. Microfluidics-assisted engineering of polymeric microcapsules with high encapsulation efficiency for protein drug delivery.

    PubMed

    Pessi, Jenni; Santos, Hélder A; Miroshnyk, Inna; JoukoYliruusi; Weitz, David A; Mirza, Sabiruddin

    2014-09-10

    In this study, microfluidic technology was employed to develop protein formulations. The microcapsules were produced with a biphasic flow to create water-oil-water (W/O/W) double emulsion droplets with ultrathin shells. Optimized microcapsule formulations containing 1% (w/w) bovine serum albumin (BSA) in the inner phase were prepared with poly(vinyl alcohol), polycaprolactone and polyethylene glycol. All the particles were found to be intact and with a particle size of 23-47 μm. Furthermore, the particles were monodisperse, non-porous and stable up to 4 weeks. The encapsulation efficiency of BSA in the microcapsules was 84%. The microcapsules released 30% of their content within 168 h. This study demonstrates that microfluidics is a powerful technique for engineering formulations for therapeutic proteins. PMID:24928131

  10. Ultrasensitive protein detection: a case for microfluidic magnetic bead-based assays.

    PubMed

    Tekin, H Cumhur; Gijs, Martin A M

    2013-12-21

    We review the use of magnetic micro- and nanoparticles ('magnetic beads') in microfluidic systems for ultrasensitive protein detection. During recent years magnetic beads have been used frequently in immunoassays, either as mobile substrates on which the target antigen is captured, as detection labels, or simultaneously as substrates and labels. The major part of the reviewed work has as application the detection of antibodies or disease biomarkers in serum or of biotoxins from food samples. Several of the most sensitive assays allow protein detection down to fg mL(-1) concentrations. We benchmark the performance of these microfluidic magnetic bead-based assays with the most promising earlier work and with alternative solutions. PMID:24145920

  11. Analysis of interactions between SNARE proteins using imaging ellipsometer coupled with microfluidic array

    PubMed Central

    Qi, Cai; Zhang, Hong; Liu, Li; Yang, Yinke; Kang, Tengfei; Hao, Wenxin; Jin, Gang; Jiang, Taijiao

    2014-01-01

    The soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins are small and abundant membrane-bound proteins, whose specific interactions mediate membrane fusion during cell fusion or cellular trafficking. In this study, we report the use of a label-free method, called imaging ellipsometer to analyze the interactions among three SNAREs, namely Sec22p, Ykt6p and Sso2p. The SNAREs were immobilized on the silicon wafer and then analyzed in a pairwise mode with microfluidic array, leading us to discover the interactions between Ykt6p and Sso2p, Sec22p and Sso2p. Moreover, by using the real-time function of the imaging ellipsometer, we were able to obtain their association constants (KA) of about 104 M−1. We argue that the use of imaging ellipsometer coupled with microfluidic device will deepen our understanding of the molecular mechanisms underlying membrane fusion process. PMID:24938428

  12. Analysis of proteins by CE, CIEF, and microfluidic devices with whole-column-imaging detection.

    PubMed

    Wu, Jiaqi; Wu, Xing-Zheng; Huang, Tiemin; Pawliszyn, Janusz

    2004-01-01

    The recently developed whole-column-imaging detection technique for capillary electrophoresis (CE) and capillary isoelectric focusing (CIEF), a commercial whole-column-imaged CIEF instrument and its standard operation protocol are introduced. Furthermore, new developments and applications of whole-column-imaging detection in protein-protein interaction study, in protein separation using microfluidic devices and CIEF methods without carrier ampholytes, as well as in 2D separation techniques are reviewed. Miniaturization of whole-column-imaging CIEF and axially illuminated fluorescence whole-column-imaging CIEF are also discussed. PMID:15163861

  13. Microfluidic cell sorter for use in developing red fluorescent proteins with improved photostability

    PubMed Central

    Davis, Lloyd M.; Lubbeck, Jennifer L.; Dean, Kevin M.; Palmer, Amy E.; Jimenez, Ralph

    2014-01-01

    This paper presents a novel microfluidic cytometer for mammalian cells that rapidly measures the irreversible photobleaching of red fluorescent proteins expressed within each cell and achieves high purity (>99%) selection of individual cells based on these measurements. The selection is achieved by using sub-millisecond timed control of a piezo-tilt mirror to steer a focused 1064-nm laser spot for optical gradient force switching following analysis of the fluorescence signals from passage of the cell through a series of 532-nm laser beams. In transit through each beam, the fluorescent proteins within the cell undergo conversion to dark states, but the microfluidic chip enables the cell to pass sufficiently slowly that recovery from reversible dark states occurs between beams, thereby enabling irreversible photobleaching to be quantified separately from the reversible dark-state conversion. The microfluidic platform achieves sorting of samples down to sub-millilitre volumes with minimal loss, wherein collected cells remain alive and can subsequently proliferate. The instrument provides a unique first tool for rapid selection of individual mammalian cells on the merits of photostability and is likely to form the basis of subsequent lab-on-a-chip platforms that combine photobleaching with other spectroscopic measurements for on-going research to develop advanced red fluorescent proteins by screening of genetic libraries. PMID:23636097

  14. Flexible metal patterning in glass microfluidic structures using femtosecond laser direct-write ablation followed by electroless plating

    NASA Astrophysics Data System (ADS)

    Xu, Jian; Midorikawa, Katsumi; Sugioka, Koji

    2014-03-01

    A simple and flexible technique for integrating metal micropatterns into glass microfluidic structures based on threedimensional femtosecond laser microfabrication is presented. Femtosecond laser direct writing followed by thermal treatment and successive chemical etching allows us to fabricate three-dimensional microfluidic structures such as microchannels and microreservoirs inside photosensitive glass. Then, the femtosecond laser direct-write ablation followed by electroless metal plating enables space-selective deposition of patterned metal films on desired locations of internal walls of the fabricated microfluidic structures. The developed technique is applied to integrate a metal microheater into a glass microchannel to control the temperature of liquid samples in the channel, which can be used as a microreactor for enhancement of chemical reactions.

  15. Online multi-channel microfluidic chip-mass spectrometry and its application for quantifying noncovalent protein-protein interactions.

    PubMed

    Liu, Wu; Chen, Qiushui; Lin, Xuexia; Lin, Jin-Ming

    2015-03-01

    To establish an automatic and online microfluidic chip-mass spectrometry (chip-MS) system, a device was designed and fabricated for microsampling by a hybrid capillary. The movement of the capillary was programmed by a computer to aspirate samples from different microfluidic channels in the form of microdroplets (typically tens of nanoliters in volume), which were separated by air plugs. The droplets were then directly analyzed by MS via paper spray ionization without any pretreatment. The feasibility and performance were demonstrated by a concentration gradient experiment. Furthermore, after eliminating the effect of nonuniform response factors by an internal standard method, determination of the association constant within a noncovalent protein-protein complex was successfully accomplished with the MS-based titration indicating the versatility and the potential of this novel platform for widespread applications. PMID:25597452

  16. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

    PubMed Central

    Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L.; Hallström, Björn M.; Liu, Zihe; Petranovic, Dina; Uhlén, Mathias; Joensson, Haakan N.; Andersson-Svahn, Helene; Nielsen, Jens

    2015-01-01

    There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories. PMID:26261321

  17. Multiplexed Affinity-Based Separation of Proteins and Cells Using Inertial Microfluidics

    PubMed Central

    Sarkar, Aniruddh; Hou, Han Wei; Mahan, Alison. E.; Han, Jongyoon; Alter, Galit

    2016-01-01

    Isolation of low abundance proteins or rare cells from complex mixtures, such as blood, is required for many diagnostic, therapeutic and research applications. Current affinity-based protein or cell separation methods use binary ‘bind-elute’ separations and are inefficient when applied to the isolation of multiple low-abundance proteins or cell types. We present a method for rapid and multiplexed, yet inexpensive, affinity-based isolation of both proteins and cells, using a size-coded mixture of multiple affinity-capture microbeads and an inertial microfluidic particle sorter device. In a single binding step, different targets–cells or proteins–bind to beads of different sizes, which are then sorted by flowing them through a spiral microfluidic channel. This technique performs continuous-flow, high throughput affinity-separation of milligram-scale protein samples or millions of cells in minutes after binding. We demonstrate the simultaneous isolation of multiple antibodies from serum and multiple cell types from peripheral blood mononuclear cells or whole blood. We use the technique to isolate low abundance antibodies specific to different HIV antigens and rare HIV-specific cells from blood obtained from HIV+ patients. PMID:27026280

  18. Microfluidic parallel patterning and cellular delivery of molecules with a nanofountain probe.

    PubMed

    Kang, Wonmo; McNaughton, Rebecca L; Yavari, Fazel; Minary-Jolandan, Majid; Safi, Asmahan; Espinosa, Horacio D

    2014-02-01

    This brief report describes a novel tool for microfluidic patterning of biomolecules and delivery of molecules into cells. The microdevice is based on integration of nanofountain probe (NFP) chips with packaging that creates a closed system and enables operation in liquid. The packaged NFP can be easily coupled to a micro/nano manipulator or atomic force microscope for precise position and force control. We demonstrate here the functionality of the device for continuous direct-write parallel patterning on a surface in air and in liquid. Because of the small volume of the probes (~3 pL), we can achieve flow rates as low as 1 fL/s and have dispensed liquid drops with submicron to 10 µm diameters in a liquid environment. Furthermore, we demonstrate that this microdevice can be used for delivery of molecules into single cells by transient permeabilization of the cell membrane (i.e., electroporation). The significant advantage of NFP-based electroporation compared with bulk electroporation and other transfection techniques is that it allows for precise and targeted delivery while minimizing stress to the cell. We discuss the ongoing development of the tool toward automated operation and its potential as a multifunctional device for microarray applications and time-dependent single-cell studies. PMID:23897012

  19. Electrical detection of protein biomarkers using bioactivated microfluidic channels

    PubMed Central

    Javanmard, Mehdi; Talasaz, Amirali H.; Nemat-Gorgani, Mohsen; Pease, Fabian; Ronaghi, Mostafa; Davis, Ronald W.

    2009-01-01

    Current methods used for analyzing biomarkers involve expensive and time consuming techniques like the Sandwich ELISA which require lengthy incubation times, high reagent costs, and bulky optical equipment. We have developed a technique involving the use of a micro-channel with integrated electrodes, functionalized with receptors specific to target biomarkers. We have applied our biochip to the rapid electrical detection and quantification of target protein biomarkers using protein functionalized micro-channels. We successfully demonstrate detection of anti-hCG antibody, at a concentration of 1 ng ml−1 and a dynamic range of three orders of magnitude, in less than one hour. We envision the use of this technique in a handheld device for multiplex high throughput analysis using an array of micro-channels for probing various protein biomarkers in clinically relevant samples such as human serum for cancer detection. PMID:19417910

  20. A Microfluidic Platform for Characterization of Protein—Protein Interactions

    PubMed Central

    Javanmard, Mehdi; Talasaz, Amirali H.; Nemat-Gorgani, Mohsen; Huber, David E.; Pease, Fabian; Ronaghi, Mostafa; Davis, Ronald W.

    2010-01-01

    Traditionally, expensive and time consuming techniques such as mass spectrometry and Western Blotting have been used for characterization of protein–protein interactions. In this paper, we describe the design, fabrication, and testing of a rapid and inexpensive sensor, involving the use of microelectrodes in a microchannel, which can be used for real-time electrical detection of specific interactions between proteins. We have successfully demonstrated detection of target glycoprotein–glycoprotein interactions, antigen-antibody interactions, and glycoprotein-antigen interactions. We have also demonstrated the ability of this technique to distinguish between strong and weak interactions. Using this approach, it may be possible to multiplex an array of these sensors onto a chip and probe a complex mixture for various types of interactions involving protein molecules. PMID:20467571

  1. Electrical detection of protein biomarkers using bioactivated microfluidic channels.

    PubMed

    Javanmard, Mehdi; Talasaz, Amirali H; Nemat-Gorgani, Mohsen; Pease, Fabian; Ronaghi, Mostafa; Davis, Ronald W

    2009-05-21

    Current methods used for analyzing biomarkers involve expensive and time consuming techniques like the Sandwich ELISA which require lengthy incubation times, high reagent costs, and bulky optical equipment. We have developed a technique involving the use of a micro-channel with integrated electrodes, functionalized with receptors specific to target biomarkers. We have applied our biochip to the rapid electrical detection and quantification of target protein biomarkers using protein functionalized micro-channels. We successfully demonstrate detection of anti-hCG antibody, at a concentration of 1 ng ml(-1) and a dynamic range of three orders of magnitude, in less than one hour. We envision the use of this technique in a handheld device for multiplex high throughput analysis using an array of micro-channels for probing various protein biomarkers in clinically relevant samples such as human serum for cancer detection. PMID:19417910

  2. Microfluidics-Based Selection of Red-Fluorescent Proteins with Decreased Rates of Photobleaching

    PubMed Central

    Dean, Kevin M.; Lubbeck, Jennifer L.; Davis, Lloyd M.; Regmi, Chola K.; Chapagain, Prem P.; Gerstman, Bernard S.; Jimenez, Ralph; Palmer, Amy E.

    2014-01-01

    Fluorescent proteins offer exceptional labeling specificity in living cells and organisms. Unfortunately, their photophysical properties remain far from ideal for long-term imaging of low-abundance cellular constituents, in large part because of their poor photostability. Despite widespread engineering efforts, improving the photostability of fluorescent proteins remains challenging due to lack of appropriate high-throughput selection methods. Here, we use molecular dynamics guided mutagenesis in conjunction with a recently developed microfluidic-based platform, which sorts cells based on their fluorescence photostability, to identify red fluorescent proteins with decreased photobleaching from a HeLa cell-based library. The identified mutant, named Kriek, has 2.5- and 4-fold higher photostability than its progenitor, mCherry, under widefield and confocal illumination, respectively. Furthermore, the results provide insight into mechanisms for enhancing photostability and their connections with other photophysical processes, thereby providing direction for ongoing development of fluorescent proteins with improved single-molecule and low-copy imaging capabilities. Insight, innovation, integration Fluorescent proteins enable imaging in situ, throughout the visible spectrum, with superb molecular specificity and single-molecule sensitivity. Unfortunately, when compared to leading small-molecule fluorophores (e.g., Cy3), fluorescent proteins, suffer from accelerated photobleaching and poor integrated photon output. This results from a lack of appropriate high-throughput methods for improving the photostability of fluorescent proteins, as well as a poor molecular understanding of fluorescent protein photobleaching. Here, we report the first application of a recently developed microfluidic cell-sorter to identify fluorescent proteins from a mCherry-derived library with improved photostability. The results provide insight into fluorescent protein photophysics, greatly

  3. A compact disk-like centrifugal microfluidic system for high-throughput nanoliter-scale protein crystallization screening.

    PubMed

    Li, Gang; Chen, Qiang; Li, Junjun; Hu, Xiaojian; Zhao, Jianlong

    2010-06-01

    A centrifuge-based microfluidic system has been developed that enables automated high-throughput and low-volume protein crystallizations. In this system, protein solution was automatically and accurately metered and dispensed into nanoliter-sized multiple reaction chambers, and it was mixed with various types of precipitants using a combination of capillary effect and centrifugal force. It has the advantages of simple fabrication, easy operation, and extremely low waste. To demonstrate the feasibility of this system, we constructed a chip containing 24 units and used it to perform lysozyme and cyan fluorescent protein (CyPet) crystallization trials. The results demonstrate that high-quality crystals can be grown and harvested from such a nanoliter-volume microfluidic system. Compared to other microfluidic technologies for protein crystallization, this microfluidic system allows zero waste, simple structure and convenient operation, which suggests that our microfluidic disk can be applied not only to protein crystallization, but also to the miniaturization of various biochemical reactions requiring precise nanoscale control. PMID:20459060

  4. Single step neutravidin patterning: a lithographic approach for patterning proteins.

    PubMed

    Verma, Sankalp; Belay, Mezigebu; Verma, Vivek

    2016-04-01

    Protein patterning on surfaces is studied extensively for its potential use in proteomic, nanostructures, drug delivery and sensing. Patterning of proteins at micro and nano scales is especially important not only to understand the function of patterned protein but also to study its interaction with subsequent layers of bio-molecules/cells. Micro scale protein patterning is especially difficult due to the fragile nature of proteins. The already available methods either involve complex chemistries or are specific to a few proteins. Thus, in this regard, a versatile approach to pattern proteins using neutravidin is developed. With this approach of lithography and subsequent lift-off of the photoresist, any biotinylated moiety can be patterned at micron scale resolution. Functionality of patterned neutravidin is confirmed by showing binding of biotinylated polystyrene beads and biotinylated antibodies. In addition, stronger physisorption of neutravidin on bare glass surface, as a result of acetone lift-off, helps sustain the protein layers onto the glass surface without the need of chemical immobilization. PMID:26899966

  5. A Microfluidic Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions.

    PubMed

    Herling, Therese W; O'Connell, David J; Bauer, Mikael C; Persson, Jonas; Weininger, Ulrich; Knowles, Tuomas P J; Linse, Sara

    2016-05-10

    The key steps in cellular signaling and regulatory pathways rely on reversible noncovalent protein-ligand binding, yet the equilibrium parameters for such events remain challenging to characterize and quantify in solution. Here, we demonstrate a microfluidic platform for the detection of protein-ligand interactions with an assay time on the second timescale and without the requirement for immobilization or the presence of a highly viscous matrix. Using this approach, we obtain absolute values for the electrophoretic mobilities characterizing solvated proteins and demonstrate quantitative comparison of results obtained under different solution conditions. We apply this strategy to characterize the interaction between calmodulin and creatine kinase, which we identify as a novel calmodulin target. Moreover, we explore the differential calcium ion dependence of calmodulin ligand-binding affinities, a system at the focal point of calcium-mediated cellular signaling pathways. We further explore the effect of calmodulin on creatine kinase activity and show that it is increased by the interaction between the two proteins. These findings demonstrate the potential of quantitative microfluidic techniques to characterize binding equilibria between biomolecules under native solution conditions. PMID:27166804

  6. Surface modification on microfluidic devices with 2-methacryloyloxyethyl phosphorylcholine polymers for reducing unfavorable protein adsorption.

    PubMed

    Sibarani, James; Takai, Madoka; Ishihara, Kazuhiko

    2007-01-15

    Surface modification of polymer materials for preparing microfluidic devices including poly(dimethyl siloxane) (PDMS) was investigated with phospholipids polymers such as poly(2-methacryloyloxylethyl phosphorylcholine(MPC)-co-n-butyl methacrylate) (PMB) and poly(MPC-co-2-ethylhexyl methacrylate-co-2-(N,N-dimethylamino)ethyl methacrylate) (PMED). The hydrophilicity of every surface on the polymer materials modified with these MPC polymers increased and the value of zeta-potential became close to zero. The protein adsorption on the polymer materials with and without the surface modification was evaluated using a protein mixture of human plasma fibrinogen and serum albumin. Amount of proteins adsorbed on these polymeric materials showed significant reduction by the surface modification with the MPC polymers compared to the uncoated surfaces ranging from 56 to 90%. Furthermore, we successfully prepared PDMS-based microchannel which was modified by simple coating with the PMB and PMED. The modified microchannel also revealed a significant reduction of adsorption of serum albumin. We conclude that the MPC polymers are useful for reducing unfavorable protein adsorption on microfluidic devices. PMID:17112710

  7. Wettability patterning for high-rate, pumpless fluid transport on open, non-planar microfluidic platforms.

    PubMed

    Ghosh, Aritra; Ganguly, Ranjan; Schutzius, Thomas M; Megaridis, Constantine M

    2014-05-01

    Surface tension driven transport of liquids on open substrates offers an enabling tool for open micro total analysis systems that are becoming increasingly popular for low-cost biomedical diagnostic devices. The present study uses a facile wettability patterning method to produce open microfluidic tracks that - due to their shape, surface texture and chemistry - are capable of transporting a wide range of liquid volumes (~1-500 μL) on-chip, overcoming viscous and other opposing forces (e.g., gravity) at the pertinent length scales. Small volumes are handled as individual droplets, while larger volumes require repeated droplet transport. The concept is developed and demonstrated with coatings based on TiO2 filler particles, which, when present in adequate (~80 wt.%) quantities within a hydrophobic fluoroacrylic polymer matrix, form composites that are intrinsically superhydrophobic. Such composite coatings become superhydrophilic upon exposure to UV light (390 nm). A commercial laser printer-based photo-masking approach is used on the coating for spatially selective wettability conversion from superhydrophobic to superhydrophilic. Carefully designed wedge-patterned surface tension confined tracks on the open-air devices move liquid on them without power input, even when acting against gravity. Simple designs of wettability patterning are used on versatile substrates (e.g., metals, polymers, paper) to demonstrate complex droplet handling tasks, e.g., merging, splitting and metered dispensing, some of which occur in 3-D geometries. Fluid transport rates of up to 350 μL s(-1) are attained. Applicability of the design on metal substrates allows these devices to be used also for other microscale engineering applications, e.g., water management in fuel cells. PMID:24622962

  8. Science Issues Associated with the Use of a Microfluidic Chip Designed Specifically for Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Holmes, Anna M.; Monaco, Lisa; Barnes, Cindy; Spearing, Scott; Jenkins, Andy; Johnson, Todd; Mayer, Derek; Cole, Helen

    2003-01-01

    The Iterative Biological Crystallization team in partnership with Caliper Technologies has produced a prototype microfluidic chip for batch crystallization that has been designed and tested. The chip is designed for the mixing and dispensing of up to five solutions with possible variation of the recipe being delivered to two growth wells. Developments that have led to the successful on-chip crystallization of a few model proteins have required investigative insight into many different areas, including fluid mixing dynamics, surface treatments, quantification and fidelity of reagent delivery. This presentation will encompass the ongoing studies and data accumulated toward these efforts.

  9. Patterned electrode-based amperometric gas sensor for direct nitric oxide detection within microfluidic devices.

    PubMed

    Cha, Wansik; Tung, Yi-Chung; Meyerhoff, Mark E; Takayama, Shuichi

    2010-04-15

    This article describes a thin amperometric nitric oxide (NO) sensor that can be microchannel embedded to enable direct real-time detection of NO produced by cells cultured within the microdevice. A key for achieving the thin ( approximately 1 mm) planar sensor configuration required for sensor-channel integration is the use of gold/indium-tin oxide patterned electrode directly on a porous polymer membrane (pAu/ITO) as the base working electrode. The electrochemically deposited Au-hexacyanoferrate layer on pAu/ITO is used to catalyze NO oxidation to nitrite at lower applied potentials (0.65-0.75 V vs Ag/AgCl) and stabilize current output. Furthermore, use of a gas-permeable membrane to separate internal sensor compartments from the sample phase imparts excellent NO selectivity over common interfering agents (e.g., nitrite, ascorbate, ammonia, etc.) present in culture media and biological fluids. The optimized sensor design reversibly detects NO down to the approximately 1 nM level in stirred buffer and <10 nM in flowing buffer when integrated within a polymeric microfluidic device. We demonstrate utility of the channel-embedded sensor by monitoring NO generation from macrophages cultured within non-gas-permeable microchannels, as they are stimulated with endotoxin. PMID:20329749

  10. Development-on-chip: in vitro neural tube patterning with a microfluidic device.

    PubMed

    Demers, Christopher J; Soundararajan, Prabakaran; Chennampally, Phaneendra; Cox, Gregory A; Briscoe, James; Collins, Scott D; Smith, Rosemary L

    2016-06-01

    Embryogenesis is a highly regulated process in which the precise spatial and temporal release of soluble cues directs differentiation of multipotent stem cells into discrete populations of specialized adult cell types. In the spinal cord, neural progenitor cells are directed to differentiate into adult neurons through the action of mediators released from nearby organizing centers, such as the floor plate and paraxial mesoderm. These signals combine to create spatiotemporal diffusional landscapes that precisely regulate the development of the central nervous system (CNS). Currently, in vivo and ex vivo studies of these signaling factors present some inherent ambiguity. In vitro methods are preferred for their enhanced experimental clarity but often lack the technical sophistication required for biological realism. In this article, we present a versatile microfluidic platform capable of mimicking the spatial and temporal chemical environments found in vivo during neural tube development. Simultaneous opposing and/or orthogonal gradients of developmental morphogens can be maintained, resulting in neural tube patterning analogous to that observed in vivo. PMID:27246712

  11. Development-on-chip: in vitro neural tube patterning with a microfluidic device

    PubMed Central

    Soundararajan, Prabakaran; Chennampally, Phaneendra; Cox, Gregory A.

    2016-01-01

    Embryogenesis is a highly regulated process in which the precise spatial and temporal release of soluble cues directs differentiation of multipotent stem cells into discrete populations of specialized adult cell types. In the spinal cord, neural progenitor cells are directed to differentiate into adult neurons through the action of mediators released from nearby organizing centers, such as the floor plate and paraxial mesoderm. These signals combine to create spatiotemporal diffusional landscapes that precisely regulate the development of the central nervous system (CNS). Currently, in vivo and ex vivo studies of these signaling factors present some inherent ambiguity. In vitro methods are preferred for their enhanced experimental clarity but often lack the technical sophistication required for biological realism. In this article, we present a versatile microfluidic platform capable of mimicking the spatial and temporal chemical environments found in vivo during neural tube development. Simultaneous opposing and/or orthogonal gradients of developmental morphogens can be maintained, resulting in neural tube patterning analogous to that observed in vivo. PMID:27246712

  12. Gene transfer and protein dynamics in stem cells using single cell electroporation in a microfluidic device.

    PubMed

    Valero, A; Post, J N; van Nieuwkasteele, J W; Ter Braak, P M; Kruijer, W; van den Berg, A

    2008-01-01

    There is great interest in genetic modification of bone marrow-derived mesenchymal stem cells (MSC), not only for research purposes but also for use in (autologous) patient-derived-patient-used transplantations. A major drawback of bulk methods for genetic modifications of (stem) cells, like bulk-electroporation, is its limited yield of DNA transfection (typically then 10%). This is even more limited when cells are present at very low numbers, as is the case for stem cells. Here we present an alternative technology to transfect cells with high efficiency (>75%), based on single cell electroporation in a microfluidic device. In a first experiment we show that we can successfully transport propidium iodide (PI) into single mouse myoblastic C2C12 cells. Subsequently, we show the use of this microfluidic device to perform successful electroporation of single mouse myoblastic C2C12 cells and single human MSC with vector DNA encoding a green fluorescent-erk1 fusion protein (EGFP-ERK1 (MAPK3)). Finally, we performed electroporation in combination with live imaging of protein expression and dynamics in response to extracellular stimuli, by fibroblast growth factor (FGF-2). We observed nuclear translocation of EGFP-ERK1 in both cell types within 15 min after FGF-2 stimulation. Due to the successful and promising results, we predict that microfluidic devices can be used for highly efficient small-scale 'genetic modification' of cells, and biological experimentation, offering possibilities to study cellular processes at the single cell level. Future applications might be small-scale production of cells for therapeutic application under controlled conditions. PMID:18094762

  13. Microfluidic Electrochemical Immunoarray for Ultrasensitive Detection of Two Cancer Biomarker Proteins in Serum

    PubMed Central

    Chikkaveeraiah, Bhaskara V.; Mani, Vigneshwaran; Patel, Vyomesh; Gutkind, J. Silvio; Rusling, James F.

    2011-01-01

    A microfluidic electrochemical immunoassay system for multiplexed detection of protein cancer biomarkers was fabricated using a molded polydimethylsiloxane channel and routine machined parts interfaced with a pump and sample injector. Using off-line capture of analytes by heavily-enzyme-labeled 1 μm superparamagnetic particle (MP)-antibody bioconjugates and capture antibodies attached to an 8-electrode measuring chip, simultaneous detection of cancer biomarker proteins prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum was achieved at sub-pg mL−1 levels. MPs were conjugated with ~90,000 antibodies and ~200,000 horseradish peroxidase (HRP) labels to provide efficient off-line capture and high sensitivity. Measuring electrodes feature a layer of 5 nm glutathione-decorated gold nanoparticles to attach antibodies that capture MP-analyte bioconjugates. Detection limits of 0.23 pg mL−1 for PSA and 0.30 pg mL−1 for IL-6 were obtained in diluted serum mixtures. PSA and IL-6 biomarkers were measured in serum of prostate cancer patients in total assay time 1.15 h and sensor array results gave excellent correlation with standard enzyme-linked immunosorbent assays (ELISA). These microfluidic immunosensors employing nanostructured surfaces and off-line analyte capture with heavily-labeled paramagnetic particles hold great promise for accurate, sensitive multiplexed detection of diagnostic cancer biomarkers. PMID:21632234

  14. Functionalized poly(ethylene glycol) diacrylate microgels by microfluidics: In situ peptide encapsulation for in serum selective protein detection.

    PubMed

    Celetti, Giorgia; Natale, Concetta Di; Causa, Filippo; Battista, Edmondo; Netti, Paolo A

    2016-09-01

    Polymeric microparticles represent a robustly platform for the detection of clinically relevant analytes in biological samples; they can be functionalized encapsulating a multiple types of biologics entities, enhancing their applications as a new class of colloid materials. Microfluidic offers a versatile platform for the synthesis of monodisperse and engineered microparticles. In this work, we report microfluidic synthesis of novel polymeric microparticles endowed with specific peptide due to its superior specificity for target binding in complex media. A peptide sequence was efficiently encapsulated into the polymeric network and protein binding occurred with high affinity (KD 0.1-0.4μM). Fluidic dynamics simulation was performed to optimize the production conditions for monodisperse and stable functionalized microgels. The results demonstrate the easy and fast realization, in a single step, of functionalized monodisperse microgels using droplet-microfluidic technique, and how the inclusion of the peptide within polymeric network improve both the affinity and the specificity of protein capture. PMID:27137799

  15. Protein stamping for MALDI mass spectrometry using an electrowetting-based microfluidic platform

    NASA Astrophysics Data System (ADS)

    Srinivasan, Vijay; Pamula, Vamsee K.; Paik, Phil; Fair, Richard B.

    2004-12-01

    MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry) is one of the most commonly used techniques for protein analysis. In conventional systems sample preparation is typically done in well-plates and transferred onto a MALDI target by robotic systems, which are complex, huge, expensive and slow. In this paper, we present a droplet-based microfluidic interface to transfer protein samples from a well-plate format onto a MALDI target for MS analysis. The droplets are actuated using the electrowetting phenomenon, and are immersed in silicone oil which prevents non-specific adsorption and enables the manipulation of high concentrations of proteins. Droplet transport and droplet formation were evaluated as a function of protein concentration using bovine serum albumin (BSA) as a test system. Droplet transport was possible for BSA concentrations up to 10mg/mL which is three orders of magnitude higher than previously reported results on handling proteins by electrowetting. Droplet formation from on-chip reservoirs, using only electrowetting forces and no external pressure assistance, was possible up to concentrations of 0.01mg/mL. An interface between a well-plate format and the electrowetting chip, and a scheme to passively stamp droplets onto a target substrate was then designed and tested by stamping BSA solutions. In two separate experiments 3.6fmoles and 16fmoles of BSA were stamped onto a glass slide using 0.001mg/mL and 0.01mg/mL samples respectively. A protein mixture with known constituents (ABI 4700 proteomics analyzer calibration solution) was stamped onto a MALDI plate and the individual proteins were correctly identified in the mass spectrum obtained using MALDI-TOF MS. The preliminary results establish the feasibility of using an electrowetting-based microfluidic system to handle proteins especially for protein stamping applications. The proposed system has a small footprint, is easy to control, and is very fast compared to conventional

  16. Protein immobilization on the surface of polydimethylsiloxane and polymethyl methacrylate microfluidic devices.

    PubMed

    Khnouf, Ruba; Karasneh, Dina; Albiss, Borhan Aldeen

    2016-02-01

    PDMS and PMMA are two of the most used polymers in the fabrication of lab-on-chip or microfluidic devices. In order to use these polymers in biological applications, it is sometimes essential to be able to bind biomolecules such as proteins and DNA to the surface of these materials. In this work, we have evaluated a number of processes that have been developed to bind protein to PDMS surfaces which include passive adsorption, passive adsorption with glutaraldehyde cross-linking, (3-aminopropyl) triethoxysilane functionalization followed by glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride cross-linkers. It has been shown that the latter technique--using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride--results in more than twice the bonding of protein to the surface of PDMS microchannels than proteins binding passively. We have also evaluated a few techniques that have been tested for the functionalization of PMMA microchannels where we have found that the use of polyethyleneimine (PEI) has led to the strongest protein-PMMA microchannel bond. We finally demonstrated the effect of PDMS curing methodology on protein adsorption to its surface, and showed that increased curing time is the factor that reduces passive adsorption the most. PMID:26534833

  17. A method to integrate patterned electrospun fibers with microfluidic systems to generate complex microenvironments for cell culture applications

    PubMed Central

    Wallin, Patric; Zandén, Carl; Carlberg, Björn; Hellström Erkenstam, Nina; Liu, Johan; Gold, Julie

    2012-01-01

    The properties of a cell’s microenvironment are one of the main driving forces in cellular fate processes and phenotype expression invivo. The ability to create controlled cell microenvironments invitro becomes increasingly important for studying or controlling phenotype expression in tissue engineering and drug discovery applications. This includes the capability to modify material surface properties within well-defined liquid environments in cell culture systems. One successful approach to mimic extra cellular matrix is with porous electrospun polymer fiber scaffolds, while microfluidic networks have been shown to efficiently generate spatially and temporally defined liquid microenvironments. Here, a method to integrate electrospun fibers with microfluidic networks was developed in order to form complex cell microenvironments with the capability to vary relevant parameters. Spatially defined regions of electrospun fibers of both aligned and random orientation were patterned on glass substrates that were irreversibly bonded to microfluidic networks produced in poly-dimethyl-siloxane. Concentration gradients obtained in the fiber containing channels were characterized experimentally and compared with values obtained by computational fluid dynamic simulations. Velocity and shear stress profiles, as well as vortex formation, were calculated to evaluate the influence of fiber pads on fluidic properties. The suitability of the system to support cell attachment and growth was demonstrated with a fibroblast cell line. The potential of the platform was further verified by a functional investigation of neural stem cell alignment in response to orientation of electrospun fibers versus a microfluidic generated chemoattractant gradient of stromal cell-derived factor 1 alpha. The described method is a competitive strategy to create complex microenvironments invitro that allow detailed studies on the interplay of topography, substrate surface properties, and soluble

  18. A method to integrate patterned electrospun fibers with microfluidic systems to generate complex microenvironments for cell culture applications.

    PubMed

    Wallin, Patric; Zandén, Carl; Carlberg, Björn; Hellström Erkenstam, Nina; Liu, Johan; Gold, Julie

    2012-06-01

    The properties of a cell's microenvironment are one of the main driving forces in cellular fate processes and phenotype expression invivo. The ability to create controlled cell microenvironments invitro becomes increasingly important for studying or controlling phenotype expression in tissue engineering and drug discovery applications. This includes the capability to modify material surface properties within well-defined liquid environments in cell culture systems. One successful approach to mimic extra cellular matrix is with porous electrospun polymer fiber scaffolds, while microfluidic networks have been shown to efficiently generate spatially and temporally defined liquid microenvironments. Here, a method to integrate electrospun fibers with microfluidic networks was developed in order to form complex cell microenvironments with the capability to vary relevant parameters. Spatially defined regions of electrospun fibers of both aligned and random orientation were patterned on glass substrates that were irreversibly bonded to microfluidic networks produced in poly-dimethyl-siloxane. Concentration gradients obtained in the fiber containing channels were characterized experimentally and compared with values obtained by computational fluid dynamic simulations. Velocity and shear stress profiles, as well as vortex formation, were calculated to evaluate the influence of fiber pads on fluidic properties. The suitability of the system to support cell attachment and growth was demonstrated with a fibroblast cell line. The potential of the platform was further verified by a functional investigation of neural stem cell alignment in response to orientation of electrospun fibers versus a microfluidic generated chemoattractant gradient of stromal cell-derived factor 1 alpha. The described method is a competitive strategy to create complex microenvironments invitro that allow detailed studies on the interplay of topography, substrate surface properties, and soluble

  19. Continuous-flow microfluidic printing of proteins for array-based applications including surface plasmon resonance imaging.

    PubMed

    Natarajan, Sriram; Katsamba, Phini S; Miles, Adam; Eckman, Josh; Papalia, Giuseppe A; Rich, Rebecca L; Gale, Bruce K; Myszka, David G

    2008-02-01

    Arraying proteins is often more challenging than creating oligonucleotide arrays. Protein concentration and purity can severely limit the capacity of spots created by traditional pin and ink jet printing techniques. To improve protein printing methods, we have developed a three-dimensional microfluidic system to deposit protein samples within discrete spots (250-microm squares) on a target surface. Our current technology produces a 48-spot array within a 0.5 x 1 cm target area. A chief advantage of this method is that samples may be introduced in continuous flow, which makes it possible to expose each spot to a larger volume of sample than would be possible with standard printing methods. Using Biacore Flexchip (Biacore AB) surface plasmon resonance array-based biosensor as a chip reader, we demonstrate that the microfluidic printer is capable of spotting proteins that are dilute (<0.1 microg/ml) and contain high concentrations of contaminating protein (>10,000-fold molar excess). We also show that the spots created by the microfluidic printer are more uniform and have better-defined borders than what can be achieved with pin printing. The ability to readily print proteins using continuous flow will help expand the application of protein arrays. PMID:17868635

  20. Towards microfluidic reactors for cell-free protein synthesis at the point-of-care

    SciTech Connect

    Timm, Andrea C.; Shankles, Peter G.; Foster, Carmen M.; Doktycz, Mitchel John; Retterer, Scott T.

    2015-12-22

    Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro- and nano-fluidic architectures, CFPS can be optimized for point-of care use. Here, we describe the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel reactor and feeder channels. This engineered membrane facilitates the exchange of metabolites, energy, and inhibitory species, prolonging the CFPS reaction and increasing protein yield. Membrane permeability can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. As a result, we show that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor.

  1. Towards microfluidic reactors for cell-free protein synthesis at the point-of-care

    DOE PAGESBeta

    Timm, Andrea C.; Shankles, Peter G.; Foster, Carmen M.; Doktycz, Mitchel John; Retterer, Scott T.

    2015-12-22

    Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro- and nano-fluidic architectures, CFPS can be optimized for point-of care use. Here, we describe the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel reactor and feeder channels. This engineered membrane facilitatesmore » the exchange of metabolites, energy, and inhibitory species, prolonging the CFPS reaction and increasing protein yield. Membrane permeability can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. As a result, we show that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor.« less

  2. Geometry-induced protein pattern formation

    PubMed Central

    Thalmeier, Dominik; Halatek, Jacob; Frey, Erwin

    2016-01-01

    Protein patterns are known to adapt to cell shape and serve as spatial templates that choreograph downstream processes like cell polarity or cell division. However, how can pattern-forming proteins sense and respond to the geometry of a cell, and what mechanistic principles underlie pattern formation? Current models invoke mechanisms based on dynamic instabilities arising from nonlinear interactions between proteins but neglect the influence of the spatial geometry itself. Here, we show that patterns can emerge as a direct result of adaptation to cell geometry, in the absence of dynamical instability. We present a generic reaction module that allows protein densities robustly to adapt to the symmetry of the spatial geometry. The key component is an NTPase protein that cycles between nucleotide-dependent membrane-bound and cytosolic states. For elongated cells, we find that the protein dynamics generically leads to a bipolar pattern, which vanishes as the geometry becomes spherically symmetrical. We show that such a reaction module facilitates universal adaptation to cell geometry by sensing the local ratio of membrane area to cytosolic volume. This sensing mechanism is controlled by the membrane affinities of the different states. We apply the theory to explain AtMinD bipolar patterns in Δ EcMinDE Escherichia coli. Due to its generic nature, the mechanism could also serve as a hitherto-unrecognized spatial template in many other bacterial systems. Moreover, the robustness of the mechanism enables self-organized optimization of protein patterns by evolutionary processes. Finally, the proposed module can be used to establish geometry-sensitive protein gradients in synthetic biological systems. PMID:26739566

  3. Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation

    PubMed Central

    Wen, Jian; Wilker, Erik W.; Yaffe, Michael B.; Jensen, Klavs F.

    2010-01-01

    Isoelectric Focusing (IEF) is the first step for two-dimensional (2D) gel electrophoresis and plays an important role in sample purification for proteomics. However, biases in protein size and pI resolution, as well as limitations in sample volume, gel capacity, sample loss, and experimental time, remain challenges. In order to address some of the limitations of traditional IEF, we present a microfluidic free flow IEF (FF-IEF) device for continuous protein separation into 24 fractions. The device reproducibly establishes a nearly linear pH gradient from 4 to 10. Optimized dynamic coatings of 4% poly (vinyl) alcohol (PVA) minimize peak broadening by transverse electrokinetic flows. Even though the device operates at high electric fields (up to 370V/cm) efficient cooling maintains solution temperature inside the separation channel controllably in the range 2 – 25 °C. Protein samples with a dynamic concentration range between µg/mL to mg/mL can be loaded into the micro device at a flow rate of 1 mL/hr and residence time of ~12 min. By using a protein complex of 9 proteins and 13 isoforms, we demonstrate improved separation with the FF-IEF system over traditional 2D gel electrophoresis. Device to device reproducibility is also illustrated through the efficient depletion of the albumin and hemoglobin assays. Post-device sample concentrations result in a 10 to 20-fold increase, which allow for isolation and detection of low abundance proteins. The separation of specific proteins from a whole cell lysate is demonstrated as an example. The micro device has the further benefits of retaining high molecular weight proteins, providing higher yield of protein that has a broader range in pI, and reducing experimental time compared to conventional IEF IGP gel strip approaches. PMID:20092256

  4. Toward Microfluidic Reactors for Cell-Free Protein Synthesis at the Point-of-Care.

    PubMed

    Timm, Andrea C; Shankles, Peter G; Foster, Carmen M; Doktycz, Mitchel J; Retterer, Scott T

    2016-02-10

    Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro and nanofluidic architectures, CFPS can be optimized for point-of-care use. Here, the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care, is described. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel "reactor" and "feeder" channels. This engineered membrane facilitates the exchange of metabolites, energy, and inhibitory species, and can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. It has been shown that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor. PMID:26690885

  5. Pattern Recognition of Adsorbing HP Lattice Proteins

    NASA Astrophysics Data System (ADS)

    Wilson, Matthew S.; Shi, Guangjie; Wüst, Thomas; Landau, David P.; Schmid, Friederike

    2015-03-01

    Protein adsorption is relevant in fields ranging from medicine to industry, and the qualitative behavior exhibited by course-grained models could shed insight for further research in such fields. Our study on the selective adsorption of lattice proteins utilizes the Wang-Landau algorithm to simulate the Hydrophobic-Polar (H-P) model with an efficient set of Monte Carlo moves. Each substrate is modeled as a square pattern of 9 lattice sites which attract either H or P monomers, and are located on an otherwise neutral surface. The fully enumerated set of 102 unique surfaces is simulated with each protein sequence. A collection of 27-monomer sequences is used- each of which is non-degenerate and protein-like. Thermodynamic quantities such as the specific heat and free energy are calculated from the density of states, and are used to investigate the adsorption of lattice proteins on patterned substrates. Research supported by NSF.

  6. Use of PLL-g-PEG in micro-fluidic devices for localizing selective and specific protein binding.

    PubMed

    Marie, Rodolphe; Beech, Jason P; Vörös, Janos; Tegenfeldt, Jonas O; Höök, Fredrik

    2006-11-21

    By utilizing flow-controlled PLL-g-PEG and PLL-g-PEGbiotin modification of predefined regions of a poly(dimethylsiloxane) (PDMS) micro-fluidic device, with an intentionally chosen large (approximately 1 cm2) internal surface area, we report rapid (10 min), highly localized (6 x 10(-6) cm2), and specific surface-based protein capture from a sample volume (100 microL) containing a low amount of protein (160 attomol in pure buffer and 400 attomol in serum). The design criteria for this surface modification were achieved using QCM-D (quartz crystal microbalance with energy dissipation monitoring) of serum protein adsorption onto PLL-g-PEG-modified oxidized PDMS. Equally good, or almost as good, results were obtained for oxidized SU-8, Topas, and poly(methyl metacrylate) (PMMA), demonstrating the generic potential of PLL-g-PEG for surface modification in various micro-fluidic applications. PMID:17107006

  7. Microfluidic flow cytometer for quantifying photobleaching of fluorescent proteins in cells.

    PubMed

    Lubbeck, Jennifer L; Dean, Kevin M; Ma, Hairong; Palmer, Amy E; Jimenez, Ralph

    2012-05-01

    Traditional flow cytometers are capable of rapid cellular assays on the basis of fluorescence intensity and light scatter. Microfluidic flow cytometers have largely followed the same path of technological development as their traditional counterparts; however, the significantly smaller transport distance and resulting lower cell speeds in microchannels provides for the opportunity to detect novel spectroscopic signatures based on multiple, nontemporally coincident excitation beams. Here, we characterize the design and operation of a cytometer with a three-beam, probe/bleach/probe geometry, employing HeLa suspension cells expressing fluorescent proteins. The data collection rate exceeds 20 cells/s under a range of beam intensities (5 kW to 179 kW/cm(2)). The measured percent photobleaching (ratio of fluorescence intensities excited by the first and third beams: S(beam3)/S(beam1)) partially resolves a mixture of four red fluorescent proteins in mixed samples. Photokinetic simulations are presented and demonstrate that the percent photobleaching reflects a combination of the reversible and irreversible photobleaching kinetics. By introducing a photobleaching optical signature, which complements traditional fluorescence intensity-based detection, this method adds another dimension to multichannel fluorescence cytometry and provides a means for flow-cytometry-based screening of directed libraries of fluorescent protein photobleaching. PMID:22424298

  8. Protein patterns as endpoints in environmental remediation

    SciTech Connect

    Bradley, B.; Brown, D.

    1995-12-31

    Biological endpoints can complement chemical analyses in monitoring environmental remediation. In some cases the levels of chemical detection are so low that the costs of clean-up to no detection would be prohibitive. And chemical tests do not indicate the availability of the contaminants to the biota. On the other hand many if not most biological tests lack specificity. The authors have investigated a protein expression assay to establish an endpoint for clean-up of sulfur mustard and breakdown products. Earthworms (Lumbricus terrestris) were exposed to sulfur mustard (SM), a breakdown product thiodiethanol (TDE), and ethylene glycol, the solvent for the two chemicals. Tissue from the lining of the coelomic cavity was taken from each of 6 worms in each treatment class. Soluble proteins were extracted and separated on one and two-dimensional (1D and 2D) gels. The 1 D gels showed no difference by eye but the patterns from control and solvent control worms on 2D gels differed from those of worms exposed to TDE and SM. The 1D gel data were digitized and analyzed by pattern recognition using artificial neural networks. The protein patterns under the two treatments and the two controls were learned in one set of data and successfully recognized in a second. This indicated that what was learned was useful in recognizing patterns induced by SM and TDE. Thus a possible endpoint for remediation would be the protein pattern at no effect levels of chemicals of interest.

  9. Optical coherence tomography imaging of microfluidic pattern with different refractive index contrast

    NASA Astrophysics Data System (ADS)

    Hu, Zhixiong; Hao, Bingtao; Liu, Wenli; Hong, Baoyu

    2014-11-01

    Optical coherence tomography (OCT) technology is analogous to ultrasound imaging, except that OCT employs light instead of sound. The non-invasive imaging method works by projecting light on test target and detecting the backscattering from the underlying layers. As the OCT technology is based on optical interference, the internal structural features and inhomogeneities induced by different refractive index contrast could be detected and displayed in the form of a gray scale or false color image. In this paper, a typical microfluidic device was produced and measured by a spectral domain OCT instrument. The internal dimensions of the lab-on-chip device were determined using the OCT imaging technology and were in agreement with results obtained with conventional confocal microscope. In order to study the effect of different refractive index contrast on OCT imaging, fluid with various refractive indexes was injected into the microfluidic channel respectively, and the acquired OCT images of the internal microfluidic channel were compared. The results demonstrate that optical coherence tomography could be used as a new metrology tool to determine the internal channel dimensions of lab-on-chip devices. Furthermore, the experiment results reveal the relations between the refractive index contrast and OCT image quality.

  10. Development of an aptamer-based impedimetric bioassay using microfluidic system and magnetic separation for protein detection.

    PubMed

    Wang, Yixian; Ye, Zunzhong; Ping, Jianfeng; Jing, Shunru; Ying, Yibin

    2014-09-15

    An aptamer-based impedimetric bioassay using the microfluidic system and magnetic separation was developed for the sensitive and rapid detection of protein. The microfluidic impedance device was fabricated through integrating the gold interdigitated array microelectrode into a flow cell made of polydimethylsiloxane (PDMS). Aptamer modified magnetic beads were used to capture and separate the target protein, and concentrated into a suitable volume. Then the complexes were injected into the microfluidic flow cell for impedance measurement. To demonstrate the high performance of this novel detection system, thrombin was employed as the target protein. The results showed that the impedance signals at the frequency of 90 kHz have a good linearity with the concentrations of thrombin in a range from 0.1 nM to 10nM and the detection limit is 0.01 nM. Compared with the reported impedimetric aptasensors for thrombin detection, this method possesses several advantages, such as the increasing sensitivity, improving reproducibility, reducing sample volume and assay time. All these demonstrate the proposed detection system is an alternative way to enable sensitive, rapid and specific detection of protein. PMID:24709326

  11. Fabrication of a polystyrene microfluidic chip coupled to electrospray ionization mass spectrometry for protein analysis.

    PubMed

    Hu, Xianqiao; Dong, Yuanyuan; He, Qiaohong; Chen, Hengwu; Zhu, Zhiwei

    2015-05-15

    A highly integrated polystyrene (PS) microfluidic chip coupled to electrospray ionization mass spectrometry for on-chip protein digestion and online analysis was developed. The immobilized enzymatic microreactor for on-chip protein digestion was integrated onto microchip via the novel method of region-selective UV-modification combined with glutaraldehyde-based immobilization. The micro film electric contact for applying high voltage was prepared on chips by using UV-directed electroless plating technique. A micro-tip was machined at the end of main channel, serving as the interface between microchip and mass spectrometric detector. On-chip digestion and online detection of protein was carried out by coupling the microchip with mass spectrometry (MS). The influences of methanol flow rate in side channel on the stability of spray and intensity of signals were investigated systematically. Also the influence of sample flow rate on the performance of immobilized enzymatic reactor were investigated. Stable spray was obtained at the spray voltage of 2.8-3.0kV and the methanol flow rate of 500-700nLmin(-1) with the relative standard deviation (RSD) of total ion current (TIC) less than 10%. The influence of sample flow rate on the performance of immobilized enzymatic reactor was also studied. The sequence coverage of protein identification decreased with the increase of flow rate of the sample solution. A sequence coverage of 96% was obtained with immobilized enzymatic reactor at the sample flow rate of 100nLmin(-1) with the reaction time of 8.4min. It could detect cytochrome c as low as 10μgmL(-1) with the developed system. No obvious decrease in protein digestion efficiency was observed after the chip continuously performed for 4h and stored for 15d. PMID:25864010

  12. Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction

    PubMed Central

    Abdallah, Bahige G.; Zatsepin, Nadia A.; Roy-Chowdhury, Shatabdi; Coe, Jesse; Conrad, Chelsie E.; Dörner, Katerina; Sierra, Raymond G.; Stevenson, Hilary P.; Camacho-Alanis, Fernanda; Grant, Thomas D.; Nelson, Garrett; James, Daniel; Calero, Guillermo; Wachter, Rebekka M.; Spence, John C. H.; Weierstall, Uwe; Fromme, Petra; Ros, Alexandra

    2015-01-01

    The advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10–100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles can be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ∼4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. This method will also permit an analysis

  13. Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction

    DOE PAGESBeta

    Abdallah, Bahige G.; Zatsepin, Nadia A.; Roy-Chowdhury, Shatabdi; Coe, Jesse; Conrad, Chelsie E.; Dörner, Katerina; Sierra, Raymond G.; Stevenson, Hilary P.; Camacho-Alanis, Fernanda; Grant, Thomas D.; et al

    2015-08-19

    We report that the advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10–100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles canmore » be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ~4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. Ultimately, this method

  14. Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction

    SciTech Connect

    Abdallah, Bahige G.; Zatsepin, Nadia A.; Roy-Chowdhury, Shatabdi; Coe, Jesse; Conrad, Chelsie E.; Dörner, Katerina; Sierra, Raymond G.; Stevenson, Hilary P.; Camacho-Alanis, Fernanda; Grant, Thomas D.; Nelson, Garrett; James, Daniel; Calero, Guillermo; Wachter, Rebekka M.; Spence, John C. H.; Weierstall, Uwe; Fromme, Petra; Ros, Alexandra

    2015-08-19

    We report that the advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10–100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles can be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ~4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. Ultimately, this method will also

  15. Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction.

    PubMed

    Abdallah, Bahige G; Zatsepin, Nadia A; Roy-Chowdhury, Shatabdi; Coe, Jesse; Conrad, Chelsie E; Dörner, Katerina; Sierra, Raymond G; Stevenson, Hilary P; Camacho-Alanis, Fernanda; Grant, Thomas D; Nelson, Garrett; James, Daniel; Calero, Guillermo; Wachter, Rebekka M; Spence, John C H; Weierstall, Uwe; Fromme, Petra; Ros, Alexandra

    2015-07-01

    The advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10-100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles can be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ∼4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. This method will also permit an analysis of

  16. Watching Single Enzymes and Fluorescent Proteins in Action in Solution Using a Microfluidic Trap

    NASA Astrophysics Data System (ADS)

    Goldsmith, Randall

    2012-02-01

    Observation of dynamics of single biomolecules over a prolonged time without altering the biomolecule via immobilization is achieved with a specialized microfluidic device. This device, the Anti-Brownian ELectrokinetic (ABEL) Trap, uses real-time electrokinetic feedback to cancel Brownian motion of single objects in solution. First, we use the ABEL Trap to study Allophycocyanin (APC), a photosynthetic antenna-protein and popular fluorescent probe. A complex relationship between fluorescence intensity and lifetime is observed, suggesting light-induced conformational changes and radiative and non-radiative rate fluctuations. Second, we apply the ABEL Trap to single molecules of the multi-copper enzyme blue Nitrite Reductase where a fluorescent label reports on the oxidation state of the Type I Copper. Redox cycling is observed and kinetic analysis allows extraction of the microscopic rate constants in the kinetic scheme. Evidence of a substrate-induced shift of the intramolecular electron transfer rate is seen. Taken together, these observations provide windows of unprecedented detail into the dynamics of solution-phase biomolecules.

  17. QR-on-a-chip: a computer-recognizable micro-pattern engraved microfluidic device for high-throughput image acquisition.

    PubMed

    Yun, Kyungwon; Lee, Hyunjae; Bang, Hyunwoo; Jeon, Noo Li

    2016-02-21

    This study proposes a novel way to achieve high-throughput image acquisition based on a computer-recognizable micro-pattern implemented on a microfluidic device. We integrated the QR code, a two-dimensional barcode system, onto the microfluidic device to simplify imaging of multiple ROIs (regions of interest). A standard QR code pattern was modified to arrays of cylindrical structures of polydimethylsiloxane (PDMS). Utilizing the recognition of the micro-pattern, the proposed system enables: (1) device identification, which allows referencing additional information of the device, such as device imaging sequences or the ROIs and (2) composing a coordinate system for an arbitrarily located microfluidic device with respect to the stage. Based on these functionalities, the proposed method performs one-step high-throughput imaging for data acquisition in microfluidic devices without further manual exploration and locating of the desired ROIs. In our experience, the proposed method significantly reduced the time for the preparation of an acquisition. We expect that the method will innovatively improve the prototype device data acquisition and analysis. PMID:26728124

  18. Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering

    PubMed Central

    Blackburn, Matthew C.; Petrova, Ekaterina; Correia, Bruno E.; Maerkl, Sebastian J.

    2016-01-01

    The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3–4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF–DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering. PMID:26704969

  19. Microfluidic Directed Synthesis of Alginate Nanogels with Tunable Pore Size for Efficient Protein Delivery.

    PubMed

    Bazban-Shotorbani, Salime; Dashtimoghadam, Erfan; Karkhaneh, Akbar; Hasani-Sadrabadi, Mohammad Mahdi; Jacob, Karl I

    2016-05-17

    Alginate is a biopolymer with favorable pH-sensitive properties for oral delivery of peptides and proteins. However, conventional alginate nanogels have limitations such as low encapsulation efficiency because of drug leaching during bead preparation and burst release in high pH values. These shortcomings originate from large pore size of the nanogels. In this work, we proposed an on-chip hydrodynamic flow focusing approach for synthesis of alginate nanogels with adjustable pore size to achieve fine-tunable release profile of the encapsulated bioactive agents. It is demonstrated that the microstructure of nanogels can be controlled through adjusting flow ratio and mixing time directed on microfluidic platforms consisting of cross-junction microchannels. In this study, the average pore size of alginate nanogels (i.e., average molecular weight between cross-links, Mc) was related to synthesis parameters. Mc was calculated from equations based on equilibrium swelling theory and proposed methods to modify the theory for pH-sensitive nanogels. In the equations we derived, size and compactness of nanogels are key factors, which can be adjusted by controlling the flow ratio. It was found that increase in flow ratio increases the size of nanogels and decreases their compactness. The size of on-chip generated nanogels for flow ratio of 0.02-0.2 was measured to be in the range of 68-138 nm. Moreover, a method based on the Mie theory was implemented to estimate the aggregation number (Nagg) of polymer chains inside the nanogels as an indicator of compactness. According to the size and compactness results along with equations of modified swelling theory, Mc obtained to be in the range of 0.5-0.8 kDa. The proposed method could be considered as a promising approach for efficient polypeptides encapsulation and their sustained release. PMID:26938744

  20. DETECTION OF TOPOLOGICAL PATTERNS IN PROTEIN NETWORKS.

    SciTech Connect

    MASLOV,S.SNEPPEN,K.

    2003-11-17

    property of many biological networks that was recently brought to attention of the scientific community [3, 4, 5] is an extremely broad distribution of node connectivities defined as the number of immediate neighbors of a given node in the network. While the majority of nodes have just a few edges connecting them to other nodes in the network, there exist some nodes, that we will refer to as ''hubs'', with an unusually large number of neighbors. The connectivity of the most connected hub in such a network is typically several orders of magnitude larger than the average connectivity in the network. Often the distribution of connectivities of individual nodes can be approximated by a scale-free power law form [3] in which case the network is referred to as scale-free. Among biological networks distributions of node connectivities in metabolic [4], protein interaction [5], and brain functional [6] networks can be reasonably approximated by a power law extending for several orders of magnitude. The set of connectivities of individual nodes is an example of a low-level (single-node) topological property of a network. While it answers the question about how many neighbors a given node has, it gives no information about the identity of those neighbors. It is clear that most functional properties of networks are defined at a higher topological level in the exact pattern of connections of nodes to each other. However, such multi-node connectivity patterns are rather difficult to quantify and compare between networks. In this work we concentrate on multi-node topological properties of protein networks. These networks (as any other biological networks) lack the top-down design. Instead, selective forces of biological evolution shape them from raw material provided by random events such as mutations within individual genes, and gene duplications. As a result their connections are characterized by a large degree of randomness. One may wonder which connectivity patterns are indeed

  1. An in-line spectrophotometer on a centrifugal microfluidic platform for real-time protein determination and calibration.

    PubMed

    Ding, Zhaoxiong; Zhang, Dongying; Wang, Guanghui; Tang, Minghui; Dong, Yumin; Zhang, Yixin; Ho, Ho-Pui; Zhang, Xuping

    2016-09-21

    In this paper, an in-line, low-cost, miniature and portable spectrophotometric detection system is presented and used for fast protein determination and calibration in centrifugal microfluidics. Our portable detection system is configured with paired emitter and detector diodes (PEDD), where the light beam between both LEDs is collimated with enhanced system tolerance. It is the first time that a physical model of PEDD is clearly presented, which could be modelled as a photosensitive RC oscillator. A portable centrifugal microfluidic system that contains a wireless port in real-time communication with a smartphone has been built to show that PEDD is an effective strategy for conducting rapid protein bioassays with detection performance comparable to that of a UV-vis spectrophotometer. The choice of centrifugal microfluidics offers the unique benefits of highly parallel fluidic actuation at high accuracy while there is no need for a pump, as inertial forces are present within the entire spinning disc and accurately controlled by varying the spinning speed. As a demonstration experiment, we have conducted the Bradford assay for bovine serum albumin (BSA) concentration calibration from 0 to 2 mg mL(-1). Moreover, a novel centrifugal disc with a spiral microchannel is proposed for automatic distribution and metering of the sample to all the parallel reactions at one time. The reported lab-on-a-disc scheme with PEDD detection may offer a solution for high-throughput assays, such as protein density calibration, drug screening and drug solubility measurement that require the handling of a large number of reactions in parallel. PMID:27531134

  2. Versatile multiple protein nanopatterning within a microfluidic channel for cell recruitment studies.

    PubMed

    Andersen, A S; Zheng, W F; Sutherland, D S; Jiang, X Y

    2015-12-21

    A novel approach combining self-assembly-based colloidal lithography and polydimethylsiloxane (PDMS) micromolding to generate complex protein nanopatterns for studying the mechanisms of leukocyte extravasation within microchannels is presented. Nanostructured surfaces sealed onto PDMS-molded microchannels are chemically functionalized in situ in an all-aqueous process to generate bi-functional chemical nanopatterns. Subsequent co-immobilization with proteins makes use of common non-covalent coupling (e.g. HIS-tags, FC-tags and biotin-tags), giving nanopatterns of arbitrary combinations of oriented, functional proteins. Up to three different proteins were simultaneously co-immobilized into the microchannel with nanoscale precision, demonstrating the complex patterns. As a proof-of-principle, a mimic of an inflamed endothelium was constructed using a macro- and nanoscale pattern of intercellular adhesion molecule 1 (ICAM1) and P-selectin, and the response of leukocytes through live cell imaging was measured. A clear result on the rolling behavior of the cells was observed with rolling limited to areas where ICAM1 and P-selectin are present. This micro/nano-interface will open new doors to investigations of how spatial distributions of proteins control cellular activity. PMID:26527486

  3. Protein assembly onto patterned microfabricated devices through enzymatic activation of fusion pro-tag.

    PubMed

    Lewandowski, Angela T; Yi, Hyunmin; Luo, Xiaolong; Payne, Gregory F; Ghodssi, Reza; Rubloff, Gary W; Bentley, William E

    2008-02-15

    We report a versatile approach for covalent surface-assembly of proteins onto selected electrode patterns of pre-fabricated devices. Our approach is based on electro-assembly of the aminopolysaccharide chitosan scaffold as a stable thin film onto patterned conductive surfaces of the device, which is followed by covalent assembly of the target protein onto the scaffold surface upon enzymatic activation of the protein's "pro-tag." For our demonstration, the model target protein is green fluorescent protein (GFP) genetically fused with a pentatyrosine pro-tag at its C-terminus, which assembles onto both two-dimensional chips and within fully packaged microfluidic devices in situ and under flow. Our surface-assembly approach enables spatial selectivity and orientational control under mild experimental conditions. We believe that our integrated approach harnessing genetic manipulation, in situ enzymatic activation, and electro-assembly makes it advantageous for a wide variety of bioMEMS and biosensing applications that require facile "biofunctionalization" of microfabricated devices. PMID:17625789

  4. Determination of cell metabolite VEGF₁₆₅ and dynamic analysis of protein-DNA interactions by combination of microfluidic technique and luminescent switch-on probe.

    PubMed

    Lin, Xuexia; Leung, Ka-Ho; Lin, Ling; Lin, Luyao; Lin, Sheng; Leung, Chung-Hang; Ma, Dik-Lung; Lin, Jin-Ming

    2016-05-15

    In this paper, we rationally design a novel G-quadruplex-selective luminescent iridium (III) complex for rapid detection of oligonucleotide and VEGF165 in microfluidics. This new probe is applied as a convenient biosensor for label-free quantitative analysis of VEGF165 protein from cell metabolism, as well as for studying the kinetics of the aptamer-protein interaction combination with a microfluidic platform. As a result, we have successfully established a quantitative analysis of VEGF165 from cell metabolism. Furthermore, based on the principles of hydrodynamic focusing and diffusive mixing, different transient states during kinetics process were monitored and recorded. Thus, the combination of microfluidic technique and G-quadruplex luminescent probe will be potentially applied in the studies of intramolecular interactions and molecule recognition in the future. PMID:26686922

  5. Automated high-throughput dense matrix protein folding screen using a liquid handling robot combined with microfluidic capillary electrophoresis.

    PubMed

    An, Philip; Winters, Dwight; Walker, Kenneth W

    2016-04-01

    Modern molecular genetics technology has made it possible to swiftly sequence, clone and mass-produce recombinant DNA for the purpose of expressing heterologous genes of interest; however, recombinant protein production systems have struggled to keep pace. Mammalian expression systems are typically favored for their ability to produce and secrete proteins in their native state, but bacterial systems benefit from rapid cell line development and robust growth. The primary drawback to prokaryotic expression systems are that recombinant proteins are generally not secreted at high levels or correctly folded, and are often insoluble, necessitating post-expression protein folding to obtain the active product. In order to harness the advantages of prokaryotic expression, high-throughput methods for executing protein folding screens and the subsequent analytics to identify lead conditions are required. Both of these tasks can be accomplished using a Biomek 3000 liquid handling robot to prepare the folding screen and to subsequently prepare the reactions for assessment using Caliper microfluidic capillary electrophoresis. By augmenting a protein folding screen with automation, the primary disadvantage of Escherichia coli expression has been mitigated, namely the labor intensive identification of the required protein folding conditions. Furthermore, a rigorous, quantitative method for identifying optimal protein folding buffer aids in the rapid development of an optimal production process. PMID:26678961

  6. Microfluidic device, and related methods

    NASA Technical Reports Server (NTRS)

    Wong, Eric W. (Inventor)

    2010-01-01

    A method of making a microfluidic device is provided. The method features patterning a permeable wall on a substrate, and surrounding the permeable wall with a solid, non-permeable boundary structure to establish a microfluidic channel having a cross-sectional dimension less than 5,000 microns and a cross-sectional area at least partially filled with the permeable wall so that fluid flowing through the microfluidic channel at least partially passes through the permeable wall.

  7. Microfluidic large-scale integration.

    PubMed

    Thorsen, Todd; Maerkl, Sebastian J; Quake, Stephen R

    2002-10-18

    We developed high-density microfluidic chips that contain plumbing networks with thousands of micromechanical valves and hundreds of individually addressable chambers. These fluidic devices are analogous to electronic integrated circuits fabricated using large-scale integration. A key component of these networks is the fluidic multiplexor, which is a combinatorial array of binary valve patterns that exponentially increases the processing power of a network by allowing complex fluid manipulations with a minimal number of inputs. We used these integrated microfluidic networks to construct the microfluidic analog of a comparator array and a microfluidic memory storage device whose behavior resembles random-access memory. PMID:12351675

  8. Simple Host−Guest Chemistry To Modulate the Process of Concentration and Crystallization of Membrane Proteins by Detergent Capture in a Microfluidic Device

    SciTech Connect

    Li, Liang; Nachtergaele, Sigrid; Seddon, Annela M.; Tereshko, Valentina; Ponomarenko, Nina; Ismagilov, Rustem F.

    2009-01-15

    This paper utilizes cyclodextrin-based host-guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-{beta}-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way.

  9. Suspended microfluidics

    PubMed Central

    Casavant, Benjamin P.; Berthier, Erwin; Theberge, Ashleigh B.; Berthier, Jean; Montanez-Sauri, Sara I.; Bischel, Lauren L.; Brakke, Kenneth; Hedman, Curtis J.; Bushman, Wade; Keller, Nancy P.; Beebe, David J.

    2013-01-01

    Although the field of microfluidics has made significant progress in bringing new tools to address biological questions, the accessibility and adoption of microfluidics within the life sciences are still limited. Open microfluidic systems have the potential to lower the barriers to adoption, but the absence of robust design rules has hindered their use. Here, we present an open microfluidic platform, suspended microfluidics, that uses surface tension to fill and maintain a fluid in microscale structures devoid of a ceiling and floor. We developed a simple and ubiquitous model predicting fluid flow in suspended microfluidic systems and show that it encompasses many known capillary phenomena. Suspended microfluidics was used to create arrays of collagen membranes, mico Dots (μDots), in a horizontal plane separating two fluidic chambers, demonstrating a transwell platform able to discern collective or individual cellular invasion. Further, we demonstrated that μDots can also be used as a simple multiplexed 3D cellular growth platform. Using the μDot array, we probed the combined effects of soluble factors and matrix components, finding that laminin mitigates the growth suppression properties of the matrix metalloproteinase inhibitor GM6001. Based on the same fluidic principles, we created a suspended microfluidic metabolite extraction platform using a multilayer biphasic system that leverages the accessibility of open microchannels to retrieve steroids and other metabolites readily from cell culture. Suspended microfluidics brings the high degree of fluidic control and unique functionality of closed microfluidics into the highly accessible and robust platform of open microfluidics. PMID:23729815

  10. 3D imaging of flow patterns in an internally-pumped microfluidic device: redox magnetohydrodynamics and electrochemically-generated density gradients.

    PubMed

    Gao, Feng; Kreidermacher, Adam; Fritsch, Ingrid; Heyes, Colin D

    2013-05-01

    Redox magnetohydrodynamics (MHD) is a promising technique for developing new electrochemical-based microfluidic flow devices with unique capabilities, such as easily switching flow direction and adjusting flow speeds and flow patterns as well as avoiding bubble formation. However, a detailed description of all the forces involved and predicting flow patterns in confined geometries is lacking. In addition to redox-MHD, density gradients caused by the redox reactions also play important roles. Flow in these devices with small fluid volumes has mainly been characterized by following microbead motion by optical microscopy either by particle tracking velocimetry (PTV) or by processing the microbead images by particle image velocimetry (PIV) software. This approach has limitations in spatial resolution and dimensionality. Here we use fluorescence correlation spectroscopy (FCS) to quantitatively and accurately measure flow speeds and patterns in the ~5-50 μm/s range in redox-MHD-based microfluidic devices, from which 3D flow maps are obtained with a spatial resolution down to 2 μm. The 2 μm spatial resolution flow speeds map revealed detailed flow profiles during redox-MHD in which the velocity increases linearly from above the electrode and reaches a plateau across the center of the cell. By combining FCS and video-microscopy (with PTV and PIV processing approaches), we are able to quantify a vertical flow of ~10 μm/s above the electrodes as a result of density gradients caused by the redox reactions and follow convection flow patterns. Overall, combining FCS, PIV, and PTV analysis of redox-MHD is a powerful combination to more thoroughly characterize the underlying forces in these promising microfluidic devices. PMID:23537496

  11. 3D Imaging of Flow Patterns in an Internally-Pumped Microfluidic Device: Redox Magnetohydrodynamics and Electrochemically-Generated Density Gradients

    PubMed Central

    Gao, Feng; Kreidermacher, Adam; Fritsch, Ingrid; Heyes, Colin D.

    2013-01-01

    Redox magnetohydrodynamics (MHD) is a promising technique for developing new electrochemical-based microfluidic flow devices with unique capabilities, such as easily switching flow direction, adjusting flow speeds and flow patterns as well as avoiding bubble formation. However, a detailed description of all the forces involved and predicting flow patterns in confined geometries is lacking. In addition to redox-MHD, density gradients caused by the redox reactions also play important roles. Flow in these devices with small fluid volumes has mainly been characterized by following microbead motion by optical microscopy either by particle tracking velocimetry (PTV) or by processing the microbead images by particle image velocimetry (PIV) software. This approach has limitations in spatial resolution and dimensionality. Here we use fluorescence correlation spectroscopy (FCS) to quantitatively and accurately measure flow speeds and patterns in the ~5-50 μm/s range in redox-MHD-based microfluidic devices, from which 3D flow maps are obtained with a spatial resolution down to 2 μm. The 2 μm spatial resolution flow speeds map revealed detailed flow profiles during redox-MHD in which the velocity increases linearly from above the electrode, and reaches a plateau across the center of the channel. By combining FCS and video-microscopy (with PTV and PIV processing approaches), we are able to quantify a vertical flow of ~10 μm/s above the electrodes as a result of density gradients caused by the redox reactions and follow convection flow patterns. Overall, combining FCS, PIV and PTV analysis of redox-MHD is a powerful combination to more thoroughly characterize the underlying forces in these promising microfluidic devices. PMID:23537496

  12. A novel approach for precisely controlled multiple cell patterning in microfluidic chips by inkjet printing and the detection of drug metabolism and diffusion.

    PubMed

    Zhang, Jie; Chen, Fengming; He, Ziyi; Ma, Yuan; Uchiyama, Katsumi; Lin, Jin-Ming

    2016-05-10

    In this work we report the use of inkjet printing as a precise and convenient means for microscale cell patterning in microfluidic chips followed by cell co-culture, stimulation and analysis. A self-made inkjet printing device was manufactured with adjustable parameters, which was capable of multiple cell printing within biocompatible materials. Sodium alginate was used as a printing matrix for cell encapsulation, and precisely distributed cell arrays on glass slides were obtained by accurate software controlled printing. By covering a PDMS layer with the corresponding microchannels onto the cell array substrate and subsequently injecting an ion cross-linking reagent, the cells containing alginate arrays gelated immediately and were immobilized on the bottom of the microchip, which could be utilized for cell culture and analysis. HepG2 cells and U251 cells were successfully co-patterned in the microchip and used for drug metabolism and diffusion experiment to imitate the in vivo situation, as a means to ascertain the capability of the system for precise microscale cell patterning in a microchip. The prodrug tegafur was metabolized by HepG2 cells into the active anticancer compound 5-fluorouracil and this produced an adverse gradient effect on U251 cells according to the distance from the HepG2 cells. The developed approach presented a feasible way to integrate inkjet cell printing and microfluidic chips for the first time, which is proved to be capable of spatially controlled printing of multiple kinds of cells into a microchip for cell culture, stimulation and analysis, which could be applied to tissue engineering, drug testing and related areas. We envision that the approach will help significantly increase the cell patterning efficacy in microfluidic chips as well as reduce the extent of laborious experimental work. PMID:27045202

  13. Automatic disease screening method using image processing for dried blood microfluidic drop stain pattern recognition.

    PubMed

    Sikarwar, Basant S; Roy, Mukesh; Ranjan, Priya; Goyal, Ayush

    2016-07-01

    This paper examines programmed automatic recognition of infection from samples of dried stains of micro-scale drops of patient blood. This technique has the upside of being low-cost and less-intrusive and not requiring puncturing the patient with a needle for drawing blood, which is especially critical for infants and the matured. It also does not require expensive pathological blood test laboratory equipment. The method is shown in this work to be successful for ailment identification in patients suffering from tuberculosis and anaemia. Illness affects the physical properties of blood, which thus influence the samples of dried micro-scale blood drop stains. For instance, if a patient has a severe drop in platelet count, which is often the case of dengue or malaria patients, the blood's physical property of viscosity drops substantially, i.e. the blood is thinner. Thus, the blood micro-scale drop stain samples can be utilised for diagnosing maladies. This paper presents programmed automatic examination of the dried micro-scale drop blood stain designs utilising an algorithm based on pattern recognition. The samples of micro-scale blood drop stains of ordinary non-infected people are clearly recognisable as well as the samples of micro-scale blood drop stains of sick people, due to key distinguishing features. As a contextual analysis, the micro-scale blood drop stains of patients infected with tuberculosis have been contrasted with the micro-scale blood drop stains of typical normal healthy people. The paper dives into the fundamental flow mechanics behind how the samples of the dried micro-scale blood drop stain is shaped. What has been found is a thick ring like feature in the dried micro-scale blood drop stains of non-ailing people and thin shape like lines in the dried micro-scale blood drop stains of patients with anaemia or tuberculosis disease. The ring like feature at the periphery is caused by an outward stream conveying suspended particles to the edge

  14. Microfluidic electronics.

    PubMed

    Cheng, Shi; Wu, Zhigang

    2012-08-21

    Microfluidics, a field that has been well-established for several decades, has seen extensive applications in the areas of biology, chemistry, and medicine. However, it might be very hard to imagine how such soft microfluidic devices would be used in other areas, such as electronics, in which stiff, solid metals, insulators, and semiconductors have previously dominated. Very recently, things have radically changed. Taking advantage of native properties of microfluidics, advances in microfluidics-based electronics have shown great potential in numerous new appealing applications, e.g. bio-inspired devices, body-worn healthcare and medical sensing systems, and ergonomic units, in which conventional rigid, bulky electronics are facing insurmountable obstacles to fulfil the demand on comfortable user experience. Not only would the birth of microfluidic electronics contribute to both the microfluidics and electronics fields, but it may also shape the future of our daily life. Nevertheless, microfluidic electronics are still at a very early stage, and significant efforts in research and development are needed to advance this emerging field. The intention of this article is to review recent research outcomes in the field of microfluidic electronics, and address current technical challenges and issues. The outlook of future development in microfluidic electronic devices and systems, as well as new fabrication techniques, is also discussed. Moreover, the authors would like to inspire both the microfluidics and electronics communities to further exploit this newly-established field. PMID:22711057

  15. Surface acoustic wave microfluidics

    PubMed Central

    Ding, Xiaoyun; Li, Peng; Lin, Sz-Chin Steven; Stratton, Zackary S.; Nama, Nitesh; Guo, Feng; Slotcavage, Daniel; Mao, Xiaole; Shi, Jinjie; Costanzo, Francesco; Huang, Tony Jun

    2014-01-01

    The recent introduction of surface acoustic wave (SAW) technology onto lab-on-a-chip platforms has opened a new frontier in microfluidics. The advantages provided by such SAW microfluidics are numerous: simple fabrication, high biocompatibility, fast fluid actuation, versatility, compact and inexpensive devices and accessories, contact-free particle manipulation, and compatibility with other microfluidic components. We believe that these advantages enable SAW microfluidics to play a significant role in a variety of applications in biology, chemistry, engineering, and medicine. In this review article, we discuss the theory underpinning SAWs and their interactions with particles and the contacting fluids in which they are suspended. We then review the SAW-enabled microfluidic devices demonstrated to date, starting with devices that accomplish fluid mixing and transport through the use of travelling SAW; we follow that by reviewing the more recent innovations achieved with standing SAW that enable such actions as particle/cell focusing, sorting, and patterning. Finally, we look forward and appraise where the discipline of SAW microfluidics could go next. PMID:23900527

  16. Specific antibody immobilization with biotin-poly(L-lysine)-g-poly(ethylene glycol) and protein A on microfluidic chips.

    PubMed

    Wen, Xiufang; He, Hongyan; Lee, L James

    2009-10-31

    Highly efficient antibody immobilization is crucial for conducting high-performance immunoassays such as enzyme-linked immunosorbent assay (ELISA) in microarray and microfluidic biochips. In this study, a biotin-poly(L-lysine)-g-poly(ethylene glycol) (biotin-PLL-g-PEG) and protein A-based technique was developed to immobilize antibody on the surface of poly(methyl methacrylate) (PMMA) microchannels. First, PMMA surface was activated by oxygen plasma, followed by poly(acrylic acid) (PAA) grafting to add functional carboxyl group for subsequent binding. After the biotin-PLL-g-PEG molecules reacted with carboxyl groups through the electrostatic interactions, biotinylated protein A was immobilized on the surface through a linking molecule, neutravidin. To evaluate the applicability of this novel immobilization strategy, human interferon-gamma (IFN-gamma) was used as a model protein. Since protein A could better control the immobilization orientation, and the combination of biotin-PLL-g-PEG and PLL-g-PEG could adjust the conformation of antibodies, antigen capture efficiency and detection signals were significantly improved on the microchips by using this strategy. The optimal grafting conditions were also experimentally determined: the biotin grafting ratio of 0.189 in the PLL-g-PEG molecule and the mixture ratio of 85% (biotin-PLL-g-PEG to PLL-g-PEG). This surface modification can be applied for targeted drug delivery, biosensor and other immunoassay applications. PMID:19647744

  17. Control of crystal polymorph in microfluidics using molluscan 28 kDa Ca²(+)-binding protein.

    PubMed

    Ji, Bozhi; Cusack, Maggie; Freer, Andy; Dobson, Phil S; Gadegaard, Nikolaj; Yin, Huabing

    2010-10-01

    Biominerals produced by biological systems in physiologically relevant environments possess extraordinary properties that are often difficult to replicate under laboratory conditions. Understanding the mechanism that underlies the process of biomineralisation can lead to novel strategies in the development of advanced materials. Using microfluidics, we have demonstrated for the first time, that an extrapallial (EP) 28 kDa protein, located in the extrapallial compartment between mantle and shell of Mytilus edulis, can influence, at both micro- and nanoscopic levels, the morphology, structure and polymorph that is laid down in the shell ultrastructure. Crucially, this influence is predominantly dependent on the existence of an EP protein concentration gradient and its consecutive interaction with Ca²(+) ions. Novel lemon-shaped hollow vaterite structures with a clearly defined nanogranular assembly occur only where particular EP protein and Ca²(+) gradients co-exist. Computational fluid dynamics enabled the progress of the reaction to be mapped and the influence of concentration gradients across the device to be calculated. Importantly, these findings could not have been observed using conventional bulk mixing methods. Our findings not only provide direct experimental evidence of the potential influence of EP proteins in crystal formation, but also offer a new biomimetic strategy to develop functional biomaterials for applications such as encapsulation and drug delivery. PMID:20820629

  18. Electro-Microfluidic Packaging

    SciTech Connect

    BENAVIDES, GILBERT L.; GALAMBOS, PAUL C.

    2002-06-01

    Electro-microfluidics is experiencing explosive growth in new product developments. There are many commercial applications for electro-microfluidic devices such as chemical sensors, biological sensors, and drop ejectors for both printing and chemical analysis. The number of silicon surface micromachined electro-microfluidic products is likely to increase. Manufacturing efficiency and integration of microfluidics with electronics will become important. Surface micromachined microfluidic devices are manufactured with the same tools as IC's (integrated circuits) and their fabrication can be incorporated into the IC fabrication process. In order to realize applications for devices must be developed. An Electro-Microfluidic Dual In-line Package (EMDIP{trademark}) was developed surface micromachined electro-microfluidic devices, a practical method for getting fluid into these to be a standard solution that allows for both the electrical and the fluidic connections needed to operate a great variety of electro-microfluidic devices. The EMDIP{trademark} includes a fan-out manifold that, on one side, mates directly with the 200 micron diameter Bosch etched holes found on the device, and, on the other side, mates to lager 1 mm diameter holes. To minimize cost the EMDIP{trademark} can be injection molded in a great variety of thermoplastics which also serve to optimize fluid compatibility. The EMDIP{trademark} plugs directly into a fluidic printed wiring board using a standard dual in-line package pattern for the electrical connections and having a grid of multiple 1 mm diameter fluidic connections to mate to the underside of the EMDIP{trademark}.

  19. Microfluidic fuel cells

    NASA Astrophysics Data System (ADS)

    Kjeang, Erik

    Microfluidic fuel cell architectures are presented in this thesis. This work represents the mechanical and microfluidic portion of a microfluidic biofuel cell project. While the microfluidic fuel cells developed here are targeted to eventual integration with biocatalysts, the contributions of this thesis have more general applicability. The cell architectures are developed and evaluated based on conventional non-biological electrocatalysts. The fuel cells employ co-laminar flow of fuel and oxidant streams that do not require a membrane for physical separation, and comprise carbon or gold electrodes compatible with most enzyme immobilization schemes developed to date. The demonstrated microfluidic fuel cell architectures include the following: a single cell with planar gold electrodes and a grooved channel architecture that accommodates gaseous product evolution while preventing crossover effects; a single cell with planar carbon electrodes based on graphite rods; a three-dimensional hexagonal array cell based on multiple graphite rod electrodes with unique scale-up opportunities; a single cell with porous carbon electrodes that provides enhanced power output mainly attributed to the increased active area; a single cell with flow-through porous carbon electrodes that provides improved performance and overall energy conversion efficiency; and a single cell with flow-through porous gold electrodes with similar capabilities and reduced ohmic resistance. As compared to previous results, the microfluidic fuel cells developed in this work show improved fuel cell performance (both in terms of power density and efficiency). In addition, this dissertation includes the development of an integrated electrochemical velocimetry approach for microfluidic devices, and a computational modeling study of strategic enzyme patterning for microfluidic biofuel cells with consecutive reactions.

  20. Wax-bonding 3D microfluidic chips.

    PubMed

    Gong, Xiuqing; Yi, Xin; Xiao, Kang; Li, Shunbo; Kodzius, Rimantas; Qin, Jianhua; Wen, Weijia

    2010-10-01

    We report a simple, low-cost and detachable microfluidic chip incorporating easily accessible paper, glass slides or other polymer films as the chip materials along with adhesive wax as the recycling bonding material. We use a laser to cut through the paper or film to form patterns and then sandwich the paper and film between glass sheets or polymer membranes. The hot-melt adhesive wax can realize bridge bonding between various materials, for example, paper, polymethylmethacrylate (PMMA) film, glass sheets, or metal plate. The bonding process is reversible and the wax is reusable through a melting and cooling process. With this process, a three-dimensional (3D) microfluidic chip is achievable by vacuating and venting the chip in a hot-water bath. To study the biocompatibility and applicability of the wax-based microfluidic chip, we tested the PCR compatibility with the chip materials first. Then we applied the wax-paper based microfluidic chip to HeLa cell electroporation (EP). Subsequently, a prototype of a 5-layer 3D chip was fabricated by multilayer wax bonding. To check the sealing ability and the durability of the chip, green fluorescence protein (GFP) recombinant Escherichia coli (E. coli) bacteria were cultured, with which the chemotaxis of E. coli was studied in order to determine the influence of antibiotic ciprofloxacin concentration on the E. coli migration. PMID:20689865

  1. Spectroscopic Analysis of Red Fluorescent Proteins and Development of a Microfluidic Cell Sorter for the Generation of Improved Variants

    NASA Astrophysics Data System (ADS)

    Lubbeck, Jennifer L.

    The discovery of the green fluorescent protein (GFP) launched the development of a wide variety of fluorescent protein (FP) mutants whose spectral and photophysical diversity revolutionized in vivo imaging. The excitation and emission spectra of red fluorescent proteins (RFPs), in particular, have been ideally tuned to a window optically favorable for in vivo work. However, their quantum yields, photostabilities and fluorescence intermittency properties require improvement if they are to be broadly employed for low-copy or single-molecule measurements. Attempts to engineer improved RFPs often result in optimization of one photophysical property at the expense of others. We developed a microfluidic-based cytometer for screening HeLa cell-based genetic RFP-libraries simultaneously on the basis of fluorescence lifetime (a proxy for quantum yield), photostability, and brightness. Ten 532 nm excitation beams interrogate each cell in flow. The first is electro-optically modulated (30 MHz) to enable lifetime measurement with phase fluorimetry. The remaining beams act as a pulse sequence for isolating the irreversible photobleaching time constant. Optical-force switching is employed to sort cells based on any combination of the photophysical parameters. Screening with this instrument enables identification of regions of the structure that synergistically affect quantum yield and photostability and the sorting capability provides a new tool for accelerating the development of next generation RFPs.

  2. Fabrication of a gel particle array in a microfluidic device for bioassays of protein and glucose in human urine samples.

    PubMed

    Lin, Ling; Gao, Zhaoxin; Wei, Huibin; Li, Haifang; Wang, Feng; Lin, Jin-Ming

    2011-09-01

    This paper describes a simple method for fabricating a series of poly(ethylene glycol) diacrylate (PEG-DA) hydrogel microstructures inside microfluidic channels as probe for proteins and glucose. In order to demonstrate the feasibility of this newly developed system, bovine serum albumin (BSA) was chosen as a model protein. PEG microcolumns were used for the parallel detection of multiple components. Using tetrabromophenol blue (TBPB) and the horseradish peroxidase/glucose oxidase reaction system, bovine serum albumin (BSA) and glucose in human urine were detected by color changes. The color changes for BSA within a concentration range of 1-150 μM, and glucose within a range of 50 mM-2 M could be directly distinguished by eyes or precisely identified by optical microscope. To show the practicability of the gel particle array, protein and glucose concentrations of real human urine samples were determined, resulting in a good correlation with hospital analysis. Notably, only a 5 µL sample was needed for a parallel measurement of both analytes. Conveniently, no special readout equipment or power source was required during the diagnosis process, which is promising for an application in rapid point-of-care diagnosis. PMID:22662039

  3. Microfluidic Diffusion Analysis of the Sizes and Interactions of Proteins under Native Solution Conditions.

    PubMed

    Arosio, Paolo; Müller, Thomas; Rajah, Luke; Yates, Emma V; Aprile, Francesco A; Zhang, Yingbo; Cohen, Samuel I A; White, Duncan A; Herling, Therese W; De Genst, Erwin J; Linse, Sara; Vendruscolo, Michele; Dobson, Christopher M; Knowles, Tuomas P J

    2016-01-26

    Characterizing the sizes and interactions of macromolecules under native conditions is a challenging problem in many areas of molecular sciences, which fundamentally arises from the polydisperse nature of biomolecular mixtures. Here, we describe a microfluidic platform for diffusional sizing based on monitoring micron-scale mass transport simultaneously in space and time. We show that the global analysis of such combined space-time data enables the hydrodynamic radii of individual species within mixtures to be determined directly by deconvoluting average signals into the contributions from the individual species. We demonstrate that the ability to perform rapid noninvasive sizing allows this method to be used to characterize interactions between biomolecules under native conditions. We illustrate the potential of the technique by implementing a single-step quantitative immunoassay that operates on a time scale of seconds and detects specific interactions between biomolecules within complex mixtures. PMID:26678709

  4. A Plug-Based Microfluidic System for Dispensing Lipidic Cubic Phase (LCP) Material Validated by Crystallizing Membrane Proteins in Lipidic Mesophases

    PubMed Central

    Li, Liang; Fu, Qiang; Kors, Christopher A.; Stewart, Lance; Nollert, Peter; Laible, Philip D.; Ismagilov, Rustem F.

    2009-01-01

    This paper presents a plug-based microfluidic system to dispense nanoliter-volume plugs of Lipidic Cubic Phase (LCP) material and subsequently merge the LCP plugs with aqueous plugs. This system was validated by crystallizing membrane proteins in lipidic mesophases, including LCP. This system allows for accurate dispensing of LCP material in nanoliter volumes, prevents inadvertent phase transitions that may occur due to dehydration by enclosing LCP in plugs, and is compatible with the traditional method of forming LCP material using a membrane protein sample, as shown by the successful crystallization of bacteriorhodopsin from Halobacterium salinarum. Conditions for the formation of LCP plugs were characterized and presented in a phase diagram. This system was also implemented using two different methods of introducing the membrane protein: 1) the traditional method of generating the LCP material using a membrane protein sample and 2) Post LCP-formation Incorporation (PLI), which involves making LCP material without protein, adding the membrane protein sample externally to the LCP material, and allowing the protein to diffuse into the LCP material or into other lipidic mesophases that may result from phase transitions. Crystals of bacterial photosynthetic reaction centers from Rhodobacter sphaeroides and Blastochloris viridis were obtained using PLI. The plug-based, LCP-assisted microfluidic system, combined with the PLI method for introducing membrane protein into LCP, should be useful for minimizing consumption of samples and broadening the screening of parameter space in membrane protein crystallization. PMID:20473353

  5. Microfluidics experiments of dissolution in a fracture. Influence of Damköhler and Péclet numbers, and of the geometry on the dissolution pattern

    NASA Astrophysics Data System (ADS)

    Osselin, Florian; Budek, Agnieszka; Cybulski, Olgierd; Szymczak, Piotr

    2015-04-01

    Dissolution of natural rocks is an ever present phenomenon in nature. The shaping of natural landscapes by the dissolution of limestone gives for example birth to exceptional features like karsts. Currently dissolution is also at the heart of key research topics as Carbon Capture and Storage or Enhanced Oil Recovery. The basics principles of dissolution are well-known, however, the sheer amount of different patterns arising from these mechanisms and the strong dependency on parameters such as pore network, chemical composition and flow rate, make it particularly difficult to study theoretically and experimentally. In this study we present a microfluidic experiment simulating the behavior of a dissolving fluid in a fracture. The experiments consist of a chip of gyspum inserted between two polycarbonate plates and subjected to a constant flow rate of pure water. The point in using microfluidics is that it allows a complete control on the experimental parameters such as geometry and chemical composition of the porous medium, flow rate, fracture aperture, roughness of the fracture walls, and an in situ observation of the geometry evolution which is impossible with 3D natural rocks. Thanks to our experiments we have been able to cover the whole range of dissolution patterns, from wormholing or DLA fingering to homogeneous dissolution, by changing Péclet and Damköhler numbers. Moreover, we have been able to tweak the geometry of our artificial fracture, inserting finger seeds or non-dissolvable obstacles. The comparison of the experimental patterns with the numerical dissolution code dissol (Szymczak and Ladd 2011) has then shown a very good correlation of the patterns, giving confidence in both experiments and modeling.

  6. Directed self-assembly of proteins into discrete radial patterns

    PubMed Central

    Thakur, Garima; Prashanthi, Kovur; Thundat, Thomas

    2013-01-01

    Unlike physical patterning of materials at nanometer scale, manipulating soft matter such as biomolecules into patterns is still in its infancy. Self-assembled monolayer (SAM) with surface density gradient has the capability to drive biomolecules in specific directions to create hierarchical and discrete structures. Here, we report on a two-step process of self-assembly of the human serum albumin (HSA) protein into discrete ring structures based on density gradient of SAM. The methodology involves first creating a 2-dimensional (2D) polyethylene glycol (PEG) islands with responsive carboxyl functionalities. Incubation of proteins on such pre-patterned surfaces results in direct self-assembly of protein molecules around PEG islands. Immobilization and adsorption of protein on such structures over time evolve into the self-assembled patterns. PMID:23719678

  7. Selective memory generalization by spatial patterning of protein synthesis

    PubMed Central

    O’Donnell, Cian; Sejnowski, Terrence J.

    2014-01-01

    Summary Protein synthesis is crucial for both persistent synaptic plasticity and long-term memory. De novo protein expression can be restricted to specific neurons within a population, and to specific dendrites within a single neuron. Despite its ubiquity, the functional benefits of spatial protein regulation for learning are unknown. We used computational modeling to study this problem. We found that spatially patterned protein synthesis can enable selective consolidation of some memories but forgetting of others, even for simultaneous events that are represented by the same neural population. Key factors regulating selectivity include the functional clustering of synapses on dendrites, and the sparsity and overlap of neural activity patterns at the circuit level. Based on these findings we proposed a novel two-step model for selective memory generalization during REM and slow-wave sleep. The pattern-matching framework we propose may be broadly applicable to spatial protein signaling throughout cortex and hippocampus. PMID:24742462

  8. Elastohydrodynamics and Kinetics of Protein Patterning in the Immunological Synapse

    PubMed Central

    Carlson, Andreas; Mahadevan, L.

    2015-01-01

    We propose a minimal mathematical model for the physical basis of membrane protein patterning in the immunological synapse (IS), which encompass membrane mechanics, protein binding kinetics and motion, and fluid flow in the synaptic cleft. Our theory leads to simple predictions for the spatial and temporal scales of protein cluster formation, growth and arrest as a function of membrane stiffness, rigidity and kinetics of the adhesive proteins, and the fluid flow in the synaptic cleft. Numerical simulations complement these scaling laws by quantifying the nucleation, growth and stabilization of proteins domains on the size of the cell. Direct comparison with experiment shows that passive elastohydrodynamics and kinetics of protein binding in the synaptic cleft can describe the short-time formation and organization of protein clusters, without evoking any active processes in the cytoskeleton. Despite the apparent complexity of the process, our analysis shows that just two dimensionless parameters characterize the spatial and temporal evolution of the protein pattern: a ratio of membrane elasticity to protein stiffness, and the ratio of a hydrodynamic time scale for fluid flow relative to the protein binding rate. A simple phase diagram encompasses the variety of patterns that can arise. PMID:26699430

  9. Datamining protein structure databanks for crystallization patterns of proteins.

    PubMed

    Valafar, Homayoun; Prestegard, James H; Valafar, Faramarz

    2002-12-01

    A study of 345 protein structures selected among 1,500 structures determined by nuclear magnetic resonance (NMR) methods, revealed useful correlations between crystallization properties and several parameters for the studied proteins. NMR methods of structure determination do not require the growth of protein crystals, and hence allow comparison of properties of proteins that have or have not been the subject of crystallographic approaches. One- and two-dimensional statistical analyses of the data confirmed a hypothesized relation between the size of the molecule and its crystallization potential. Furthermore, two-dimensional Bayesian analysis revealed a significant relationship between relative ratio of different secondary structures and the likelihood of success for crystallization trials. The most immediate result is an apparent correlation of crystallization potential with protein size. Further analysis of the data revealed a relationship between the unstructured fraction of proteins and the success of its crystallization. Utilization of Bayesian analysis on the latter correlation resulted in a prediction performance of about 64%, whereas a two-dimensional Bayesian analysis succeeded with a performance of about 75%. PMID:12594078

  10. Detection of Streptavidin-Biotin Protein Complexes Using Three-Dimensional MOSFET in the Si Micro-Fluidic Channel

    NASA Astrophysics Data System (ADS)

    Han, Dae-Il; Kim, Dong-Sun; Park, Jee-Eun; Shin, Jang-Kyoo; Kong, Seong-Ho; Choi, Pyung; Lee, Jong-Hyun; Lim, Geunbae

    2005-07-01

    To detect the electrical reaction characteristics of streptavidin-biotin protein complexes, a three-dimensional (3-D) p-channel metal oxide semiconductor field effect transistor (PMOSFET)-type biosensor was fabricated in the convex corner of a micro-fluidic channel by tetramethyl ammonium hydroxide (TMAH) anisotropic etching. Au, which has a chemical affinity with thiol, was used as the gate metal for immobilizing a self-assembled monolayer (SAM). The SAM was used to immobilize streptavidin. The hydroxyl group of SAM was bound with the amine group of streptavidin. After that, streptavidin and biotin were bound by their high affinity (Ka˜ 1015 Mol-1). The measurements were performed in phosphate-buffered saline (PBS; pH 6.4, 20 mM) solution. Ag/AgCl was used as the reference electrode. The bindings of SAM, streptavidin and biotin caused a variation in the drain current of the 3-D MOSFET. To verify the interaction among the SAM, streptavidin and biotin, surface plasmon resonance (SPR) measurement was performed.

  11. High-throughput gene expression analysis at the level of single proteins using a microfluidic turbidostat and automated cell tracking

    PubMed Central

    Ullman, G.; Wallden, M.; Marklund, E. G.; Mahmutovic, A.; Razinkov, Ivan; Elf, J.

    2013-01-01

    We have developed a method combining microfluidics, time-lapsed single-molecule microscopy and automated image analysis allowing for the observation of an excess of 3000 complete cell cycles of exponentially growing Escherichia coli cells per experiment. The method makes it possible to analyse the rate of gene expression at the level of single proteins over the bacterial cell cycle. We also demonstrate that it is possible to count the number of non-specifically DNA binding LacI–Venus molecules using short excitation light pulses. The transcription factors are localized on the nucleoids in the cell and appear to be uniformly distributed on chromosomal DNA. An increase in the expression of LacI is observed at the beginning of the cell cycle, possibly because some gene copies are de-repressed as a result of partitioning inequalities at cell division. Finally, a size–growth rate uncertainty relation is observed where cells living in rich media vary more in the length at birth than in generation time, and the opposite is true for cells living in poorer media. PMID:23267179

  12. Polyacrylamide gel plugs enabling 2-D microfluidic protein separations via isoelectric focusing and multiplexed sodium dodecyl sulfate gel electrophoresis.

    PubMed

    Liu, Jikun; Yang, Shuang; Lee, Cheng S; DeVoe, Don L

    2008-06-01

    In situ photopolymerized polyacrylamide (PAAm) gel plugs are used as hydrodynamic flow control elements in a multidimensional microfluidic system combining IEF and parallel SDS gel electrophoresis for protein separations. The PAAm gel plugs offer a simple method to reduce undesirable bulk flow and limit reagent/sample crosstalk without placing unwanted constraints on the selection of separation media, and without hindering electrokinetic ion migration in the complex microchannel network. In addition to improving separation reproducibility, the discrete gel plugs integrated into critical regions of the chip enable the use of a simple pressure-driven sample injection method which avoids electrokinetic injection bias. The gel plugs also serve to greatly simplify operation of the spatially multiplexed system by eliminating the need for complex external fluidic interfaces. Using an FITC-labeled Escherichia coli cell lysate as a model system, the use of gel plugs is shown to significantly enhance separation reproducibility in a chip containing five parallel CGE channels, with an average variance in peak elution time of only 4.1%. PMID:18449857

  13. High-throughput gene expression analysis at the level of single proteins using a microfluidic turbidostat and automated cell tracking.

    PubMed

    Ullman, G; Wallden, M; Marklund, E G; Mahmutovic, A; Razinkov, Ivan; Elf, J

    2013-02-01

    We have developed a method combining microfluidics, time-lapsed single-molecule microscopy and automated image analysis allowing for the observation of an excess of 3000 complete cell cycles of exponentially growing Escherichia coli cells per experiment. The method makes it possible to analyse the rate of gene expression at the level of single proteins over the bacterial cell cycle. We also demonstrate that it is possible to count the number of non-specifically DNA binding LacI-Venus molecules using short excitation light pulses. The transcription factors are localized on the nucleoids in the cell and appear to be uniformly distributed on chromosomal DNA. An increase in the expression of LacI is observed at the beginning of the cell cycle, possibly because some gene copies are de-repressed as a result of partitioning inequalities at cell division. Finally, a size-growth rate uncertainty relation is observed where cells living in rich media vary more in the length at birth than in generation time, and the opposite is true for cells living in poorer media. PMID:23267179

  14. Expression Pattern of Id Proteins in Medulloblastoma

    PubMed Central

    Snyder, Andrew D.; Dulin-Smith, Ashley N.; Houston, Ronald H.; Durban, Ashley N.; Brisbin, Bethany J.; Oostra, Tyler D.; Marshall, Jordan T.; Kahwash, Basil M.

    2013-01-01

    Inhibitor of DNA binding or inhibitor of differentiation (Id) proteins are up regulated in a variety of neoplasms, particularly in association with high-grade, poorly differentiated tumors, while differentiated tissues show little or no Id expression. The four Id genes are members of the helix-loop-helix (HLH) family of transcription factors and act as negative regulators of transcription by binding to and sequestering HLH complexes. We tested the hypothesis that Id proteins are overexpressed in medulloblastoma by performing immunohistochemistry using a medulloblastoma tissue microarray with 45 unique medulloblastoma and 11 normal control cerebella, and antibodies specific for Id1, Id2, Id3, and Id4. A semi-quantitative staining score that took staining intensity and the proportion of immunoreactive cells into account was used. Id1 was not detected in normal cerebella or in medulloblastoma cells, but 78 % of tumors showed strong Id1 expression in endothelial nuclei of tumor vessels. Id2 expression was scant in normal cerebella and increased in medulloblastoma (median staining score: 4). Id3 expression was noted in some neurons of the developing cerebellar cortex, but it was markedly up regulated in medulloblastoma (median staining score: 12) and in tumor endothelial cells. Id4 was not expressed in normal cerebella or in tumor cells. Id2 or Id3 overexpression drove proliferation in medulloblastoma cell lines by altering the expression of critical cell cycle regulatory proteins in favor of cell proliferation. This study shows that Id1 expression in endothelial cells may contribute to angiogenic processes and that increased expression of Id2 and Id3 in medulloblastoma is potentially involved in tumor cell proliferation and survival. PMID:23397264

  15. Microtubule patterning in the presence of moving motor proteins.

    PubMed

    White, D; de Vries, G; Martin, J; Dawes, A

    2015-10-01

    Cytoskeletal polymers such as microtubules (MTs) interact with motor proteins to form higher-order structures. In vitro experiments have shown that MT patterns such as asters, bundles, and vortices can form under the influence of a single type of dynamic motor protein. MTs also can form anti-parallel bundles, similar to bundles that form the mitotic spindle during cell division, under the influence of two types of moving motors with opposite directionality. Despite the importance of MT structures, their mechanism of formation is not yet understood. We develop an integro-partial differential equation model to describe the dynamic interactions between MTs and moving motor proteins. Our model takes into account motor protein speed, processivity, density, and directionality, as well as MT treadmilling and reorganization due to interactions with motors. Simulation results show that plus-end directed motor proteins can form vortex patterns at low motor density, while minus-end directed motor proteins form aster patterns at similar densities. Also, motor proteins with opposite directionality are able to organize MTs into anti-parallel bundles. Our model is able to provide a quantitative and qualitative description of MT patterning, providing insights into possible mechanisms of spindle formation. PMID:26159812

  16. Ice matrix in reconfigurable microfluidic systems

    NASA Astrophysics Data System (ADS)

    Bossi, A. M.; Vareijka, M.; Piletska, E. V.; Turner, A. P. F.; Meglinski, I.; Piletsky, S. A.

    2013-07-01

    Microfluidic devices find many applications in biotechnologies. Here, we introduce a flexible and biocompatible microfluidic ice-based platform with tunable parameters and configuration of microfluidic patterns that can be changed multiple times during experiments. Freezing and melting of cavities, channels and complex relief structures created and maintained in the bulk of ice by continuous scanning of an infrared laser beam are used as a valve action in microfluidic systems. We demonstrate that pre-concentration of samples and transport of ions and dyes through the open channels created can be achieved in ice microfluidic patterns by IR laser-assisted zone melting. The proposed approach can be useful for performing separation and sensing processes in flexible reconfigurable microfluidic devices.

  17. Protein patterning: a comparison of direct spotting versus microcontact printing

    NASA Astrophysics Data System (ADS)

    Clancy, Kathryn F. A.; Nicolau, Dan V.

    2015-03-01

    Protein microarrays are used various research areas including drug discovery, diagnosis, and analysis of protein-ligand interactions. Their efficacy depends on a well-defined pattern of immobilized proteins that also have retained their bioactivity. Protein microarrays are classically fabricated using the robotic spotting drop method ("pin printing"), which can lead to spots with uneven protein concentration within the spotted area, leading to difficult to quantify readings. Among the alternative techniques, microcontact printing (μCP) with a poly(dimethylsiloxane) (PDMS) stamp appears to deliver more defined protein patterns on surfaces, while maintaining bioactivity for a wide range of proteins. Here we have quantitatively compared the distribution of fluorescently labeled proteins deposited using direct pipetting, pin printing and μCP printing with flat stamps onto various functionalized glass surfaces of different contact angles through fluorescent microscopy. The uniformity of the deposited protein spots across deposition techniques was also qualitatively analyzed. It was found that with the use of either the direct pipetting or pin printing techniques that protein concentration on surfaces varied largely across surfaces with different contact angles, whereas adsorption did not vary significantly when using the μCP printing Furthermore, when μCP printing was performed with flat relief structures the spot inhomogeneity was lower than when classical methods were used, and even less so when a pyramid relief structure was used. This suggests that μCP printing with pyramid relief structures could produce protein patterns on various surfaces and with increased spot uniformity to enable more reliable protein microarrays.

  18. Geometry sensing by self-organized protein patterns

    PubMed Central

    Schweizer, Jakob; Loose, Martin; Bonny, Mike; Kruse, Karsten; Mönch, Ingolf; Schwille, Petra

    2012-01-01

    In the living cell, proteins are able to organize space much larger than their dimensions. In return, changes of intracellular space can influence biochemical reactions, allowing cells to sense their size and shape. Despite the possibility to reconstitute protein self-organization with only a few purified components, we still lack knowledge of how geometrical boundaries affect spatiotemporal protein patterns. Following a minimal systems approach, we used purified proteins and photolithographically patterned membranes to study the influence of spatial confinement on the self-organization of the Min system, a spatial regulator of bacterial cytokinesis, in vitro. We found that the emerging protein pattern responds even to the lateral, two-dimensional geometry of the membrane such that, as in the three-dimensional cell, Min protein waves travel along the longest axis of the membrane patch. This shows that for spatial sensing the Min system does not need to be enclosed in a three-dimensional compartment. Using a computational model we quantitatively analyzed our experimental findings and identified persistent binding of MinE to the membrane as requirement for the Min system to sense geometry. Our results give insight into the interplay between geometrical confinement and biochemical patterns emerging from a nonlinear reaction–diffusion system. PMID:22949703

  19. Multistability and dynamic transitions of intracellular Min protein patterns.

    PubMed

    Wu, Fabai; Halatek, Jacob; Reiter, Matthias; Kingma, Enzo; Frey, Erwin; Dekker, Cees

    2016-01-01

    Cells owe their internal organization to self-organized protein patterns, which originate and adapt to growth and external stimuli via a process that is as complex as it is little understood. Here, we study the emergence, stability, and state transitions of multistable Min protein oscillation patterns in live Escherichia coli bacteria during growth up to defined large dimensions. De novo formation of patterns from homogenous starting conditions is observed and studied both experimentally and in simulations. A new theoretical approach is developed for probing pattern stability under perturbations. Quantitative experiments and simulations show that, once established, Min oscillations tolerate a large degree of intracellular heterogeneity, allowing distinctly different patterns to persist in different cells with the same geometry. Min patterns maintain their axes for hours in experiments, despite imperfections, expansion, and changes in cell shape during continuous cell growth. Transitions between multistable Min patterns are found to be rare events induced by strong intracellular perturbations. The instances of multistability studied here are the combined outcome of boundary growth and strongly nonlinear kinetics, which are characteristic of the reaction-diffusion patterns that pervade biology at many scales. PMID:27279643

  20. Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood.

    PubMed

    Park, Yang-Seok; Sunkara, Vijaya; Kim, Yubin; Lee, Won Seok; Han, Ja-Ryoung; Cho, Yoon-Kyoung

    2016-01-01

    Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins. PMID:27167836

  1. A Microfluidic Bioreactor with in Situ SERS Imaging for the Study of Controlled Flow Patterns of Biofilm Precursor Materials

    PubMed Central

    Paquet-Mercier, François; Aznaveh, Nahid Babaei; Safdar, Muhammad; Greener, Jesse

    2013-01-01

    A microfluidic bioreactor with an easy to fabricate nano-plasmonic surface is demonstrated for studies of biofilms and their precursor materials via Surface Enhanced Raman Spectroscopy (SERS). The system uses a novel design to induce sheath flow confinement of a sodium citrate biofilm precursor stream against the SERS imaging surface to measure spatial variations in the concentration profile. The unoptimised SERS enhancement was approximately 2.5 × 104, thereby improving data acquisition time, reducing laser power requirements and enabling a citrate detection limit of 0.1 mM, which was well below the concentrations used in biofilm nutrient solutions. The flow confinement was observed by both optical microscopy and SERS imaging with good complementarity. We demonstrate the new bioreactor by growing flow-templated biofilms on the microchannel wall. This work opens the way for in situ spectral imaging of biofilms and their biochemical environment under dynamic flow conditions. PMID:24172286

  2. Microfluidic bead suspension hopper.

    PubMed

    Price, Alexander K; MacConnell, Andrew B; Paegel, Brian M

    2014-05-20

    Many high-throughput analytical platforms, from next-generation DNA sequencing to drug discovery, rely on beads as carriers of molecular diversity. Microfluidic systems are ideally suited to handle and analyze such bead libraries with high precision and at minute volume scales; however, the challenge of introducing bead suspensions into devices before they sediment usually confounds microfluidic handling and analysis. We developed a bead suspension hopper that exploits sedimentation to load beads into a microfluidic droplet generator. A suspension hopper continuously delivered synthesis resin beads (17 μm diameter, 112,000 over 2.67 h) functionalized with a photolabile linker and pepstatin A into picoliter-scale droplets of an HIV-1 protease activity assay to model ultraminiaturized compound screening. Likewise, trypsinogen template DNA-coated magnetic beads (2.8 μm diameter, 176,000 over 5.5 h) were loaded into droplets of an in vitro transcription/translation system to model a protein evolution experiment. The suspension hopper should effectively remove any barriers to using suspensions as sample inputs, paving the way for microfluidic automation to replace robotic library distribution. PMID:24761972

  3. Development of Plate Reader and On-Line Microfluidic Screening to Identify Ligands of the 5-Hydroxytryptamine Binding Protein in Venoms

    PubMed Central

    Otvos, Reka A.; Krishnamoorthy Iyer, Janaki; van Elk, René; Ulens, Chris; Niessen, Wilfried M. A.; Somsen, Govert W.; Kini, R. Manjunatha; Smit, August B.; Kool, Jeroen

    2015-01-01

    The 5-HT3 receptor is a ligand-gated ion channel, which is expressed in the nervous system. Its antagonists are used clinically for treatment of postoperative- and radiotherapy-induced emesis and irritable bowel syndrome. In order to better understand the structure and function of the 5-HT3 receptor, and to allow for compound screening at this receptor, recently a serotonin binding protein (5HTBP) was engineered with the Acetylcholine Binding Protein as template. In this study, a fluorescence enhancement assay for 5HTBP ligands was developed in plate-reader format and subsequently used in an on-line microfluidic format. Both assay types were validated using an existing radioligand binding assay. The on-line microfluidic assay was coupled to HPLC via a post-column split which allowed parallel coupling to a mass spectrometer to collect MS data. This high-resolution screening (HRS) system is well suitable for compound mixture analysis. As a proof of principle, the venoms of Dendroapsis polylepis, Pseudonaja affinis and Pseudonaja inframacula snakes were screened and the accurate masses of the found bioactives were established. To demonstrate the subsequent workflow towards structural identification of bioactive proteins and peptides, the partial amino acid sequence of one of the bioactives from the Pseudonaja affinis venom was determined using a bottom-up proteomics approach. PMID:26114334

  4. Development of Plate Reader and On-Line Microfluidic Screening to Identify Ligands of the 5-Hydroxytryptamine Binding Protein in Venoms.

    PubMed

    Otvos, Reka A; Iyer, Janaki Krishnamoorthy; van Elk, René; Ulens, Chris; Niessen, Wilfried M A; Somsen, Govert W; Kini, R Manjunatha; Smit, August B; Kool, Jeroen

    2015-07-01

    The 5-HT3 receptor is a ligand-gated ion channel, which is expressed in the nervous system. Its antagonists are used clinically for treatment of postoperative- and radiotherapy-induced emesis and irritable bowel syndrome. In order to better understand the structure and function of the 5-HT3 receptor, and to allow for compound screening at this receptor, recently a serotonin binding protein (5HTBP) was engineered with the Acetylcholine Binding Protein as template. In this study, a fluorescence enhancement assay for 5HTBP ligands was developed in plate-reader format and subsequently used in an on-line microfluidic format. Both assay types were validated using an existing radioligand binding assay. The on-line microfluidic assay was coupled to HPLC via a post-column split which allowed parallel coupling to a mass spectrometer to collect MS data. This high-resolution screening (HRS) system is well suitable for compound mixture analysis. As a proof of principle, the venoms of Dendroapsis polylepis, Pseudonaja affinis and Pseudonaja inframacula snakes were screened and the accurate masses of the found bioactives were established. To demonstrate the subsequent workflow towards structural identification of bioactive proteins and peptides, the partial amino acid sequence of one of the bioactives from the Pseudonaja affinis venom was determined using a bottom-up proteomics approach. PMID:26114334

  5. Pattern recognition methods for protein functional site prediction.

    PubMed

    Yang, Zheng Rong; Wang, Lipo; Young, Natasha; Trudgian, Dave; Chou, Kuo-Chen

    2005-10-01

    Protein functional site prediction is closely related to drug design, hence to public health. In order to save the cost and the time spent on identifying the functional sites in sequenced proteins in biology laboratory, computer programs have been widely used for decades. Many of them are implemented using the state-of-the-art pattern recognition algorithms, including decision trees, neural networks and support vector machines. Although the success of this effort has been obvious, advanced and new algorithms are still under development for addressing some difficult issues. This review will go through the major stages in developing pattern recognition algorithms for protein functional site prediction and outline the future research directions in this important area. PMID:16248799

  6. Integrated microfluidic platforms for investigating neuronal networks

    NASA Astrophysics Data System (ADS)

    Kim, Hyung Joon

    This dissertation describes the development and application of integrated microfluidics-based assay platforms to study neuronal activities in the nervous system in-vitro. The assay platforms were fabricated using soft lithography and micro/nano fabrication including microfluidics, surface patterning, and nanomaterial synthesis. The use of integrated microfluidics-based assay platform allows culturing and manipulating many types of neuronal tissues in precisely controlled microenvironment. Furthermore, they provide organized multi-cellular in-vitro model, long-term monitoring with live cell imaging, and compatibility with molecular biology techniques and electrophysiology experiment. In this dissertation, the integrated microfluidics-based assay platforms are developed for investigation of neuronal activities such as local protein synthesis, impairment of axonal transport by chemical/physical variants, growth cone path finding under chemical/physical cues, and synaptic transmission in neuronal circuit. Chapter 1 describes the motivation, objectives, and scope for developing in-vitro platform to study various neuronal activities. Chapter 2 introduces microfluidic culture platform for biochemical assay with large-scale neuronal tissues that are utilized as model system in neuroscience research. Chapter 3 focuses on the investigation of impaired axonal transport by beta-Amyloid and oxidative stress. The platform allows to control neuronal processes and to quantify mitochondrial movement in various regions of axons away from applied drugs. Chapter 4 demonstrates the development of microfluidics-based growth cone turning assay to elucidate the mechanism underlying axon guidance under soluble factors and shear flow. Using this platform, the behaviors of growth cone of mammalian neurons are verified under the gradient of inhibitory molecules and also shear flow in well-controlled manner. In Chapter 5, I combine in-vitro multicellular model with microfabricated MEA

  7. Effects of shear on P-selectin deposition in microfluidic channels.

    PubMed

    Shimp, Eddie A; Alsmadi, Nesreen Z; Cheng, Tiffany; Lam, Kevin H; Lewis, Christopher S; Schmidtke, David W

    2016-03-01

    Traditional leukocyte adhesion assays have provided significant insight into the mechanisms of leukocyte rolling in part through the use of homogeneously coated surfaces. These assays typically involve protein coating of glass coverslips or plastic petri dishes applied via a static drop of protein solution. With this approach, it is difficult to spatially control the location of proteins to fabricate surface-bound protein gradients that mimic in vivo situations. Microfluidic patterning of proteins with microfluidic devices has become a popular technique due to the ability to spatially pattern proteins on a cellular scale. Despite the advantages of microfluidic patterning, few studies have systematically investigated the effects of perfusion time, protein concentration, and perfusion shear stress on protein deposition. Herein, we demonstrated the fabrication of both line and step gradients of P-selectin on glass substrates that support cell rolling and adhesion assays. Investigation of the flow conditions during the microfluidic patterning led to several significant findings. We observed that the protein deposition time of 5 min was sufficient to deposit adequate P-selectin to support neutrophil rolling. We demonstrated that the amount of membrane P-selectin (mP-selectin) or recombinant P-selectin (rP-selectin) deposited showed a dependence on the perfusion shear stress between 4.0 and 32.0 dyn/cm(2), while similar studies with fibronectin or fibrinogen showed no shear stress dependence. Finally, we also created step changes in surface adherent protein concentration of P-selectin to characterize leukocyte-rolling behavior in response to sudden changes in ligand density. PMID:27190563

  8. Photo-patterned free-standing hydrogel microarrays for massively parallel protein analysis

    NASA Astrophysics Data System (ADS)

    Duncombe, Todd A.; Herr, Amy E.

    2015-03-01

    Microfluidic technologies have largely been realized within enclosed microchannels. While powerful, a principle limitation of closed-channel microfluidics is the difficulty for sample extraction and downstream processing. To address this limitation and expand the utility of microfluidic analytical separation tools, we developed an openchannel hydrogel architecture for rapid protein analysis. Designed for compatibility with slab-gel polyacrylamide gel electrophoresis (PAGE) reagents and instruments, we detail the development of free-standing polyacrylamide gel (fsPAG) microstructures supporting electrophoretic performance rivalling that of microfluidic platforms. Owing to its open architecture - the platform can be easily interfaced with automated robotic controllers and downstream processing (e.g., sample spotters, immunological probing, mass spectroscopy). The fsPAG devices are directly photopatterened atop of and covalently attached to planar polymer or glass surfaces. Due to the fast < 1 hr design-prototype-test cycle - significantly faster than mold based fabrication techniques - rapid prototyping devices with fsPAG microstructures provides researchers a powerful tool for developing custom analytical assays. Leveraging the rapid prototyping benefits - we up-scale from a unit separation to an array of 96 concurrent fsPAGE assays in 10 min run time driven by one electrode pair. The fsPAGE platform is uniquely well-suited for massively parallelized proteomics, a major unrealized goal from bioanalytical technology.

  9. The Microfluidic Jukebox

    PubMed Central

    Tan, Say Hwa; Maes, Florine; Semin, Benoît; Vrignon, Jérémy; Baret, Jean-Christophe

    2014-01-01

    Music is a form of art interweaving people of all walks of life. Through subtle changes in frequencies, a succession of musical notes forms a melody which is capable of mesmerizing the minds of people. With the advances in technology, we are now able to generate music electronically without relying solely on physical instruments. Here, we demonstrate a musical interpretation of droplet-based microfluidics as a form of novel electronic musical instruments. Using the interplay of electric field and hydrodynamics in microfluidic devices, well controlled frequency patterns corresponding to musical tracks are generated in real time. This high-speed modulation of droplet frequency (and therefore of droplet sizes) may also provide solutions that reconciles high-throughput droplet production and the control of individual droplet at production which is needed for many biochemical or material synthesis applications. PMID:24781785

  10. The Microfluidic Jukebox

    NASA Astrophysics Data System (ADS)

    Tan, Say Hwa; Maes, Florine; Semin, Benoît; Vrignon, Jérémy; Baret, Jean-Christophe

    2014-04-01

    Music is a form of art interweaving people of all walks of life. Through subtle changes in frequencies, a succession of musical notes forms a melody which is capable of mesmerizing the minds of people. With the advances in technology, we are now able to generate music electronically without relying solely on physical instruments. Here, we demonstrate a musical interpretation of droplet-based microfluidics as a form of novel electronic musical instruments. Using the interplay of electric field and hydrodynamics in microfluidic devices, well controlled frequency patterns corresponding to musical tracks are generated in real time. This high-speed modulation of droplet frequency (and therefore of droplet sizes) may also provide solutions that reconciles high-throughput droplet production and the control of individual droplet at production which is needed for many biochemical or material synthesis applications.

  11. Digital Microfluidic Logic Gates

    NASA Astrophysics Data System (ADS)

    Zhao, Yang; Xu, Tao; Chakrabarty, Krishnendu

    Microfluidic computing is an emerging application for microfluidics technology. We propose microfluidic logic gates based on digital microfluidics. Using the principle of electrowetting-on-dielectric, AND, OR, NOT and XOR gates are implemented through basic droplet-handling operations such as transporting, merging and splitting. The same input-output interpretation enables the cascading of gates to create nontrivial computing systems. We present a potential application for microfluidic logic gates by implementing microfluidic logic operations for on-chip HIV test.

  12. Microfluidics with Gel Emulsions

    NASA Astrophysics Data System (ADS)

    Priest, Craig; Surenjav, Enkhtuul; Herminghaus, Stephan; Seemann, Ralf

    2006-03-01

    Microfluidic processing is usually achieved using single phase liquids. Instead, we use monodisperse emulsions to compartment liquids within microchannel geometries. At low continuous phase volume fractions, droplets self-organize to form well-defined arrangements, analogous to foam. While it is well-known that confined geometries can induce rearrangement of foam compartments at the millimeter-scale, similar dynamics are also expected for gel emulsions. We have studied online generation, organization and manipulation of gel emulsions using a variety of microchannel geometries. ``Passive'' reorganization, based on fixed channel geometries, can be supplemented by ``active'' manipulation by incorporating a ferrofluid phase. A ferromagnetic phase facilitates reorganization of liquid compartments on demand using an electromagnetic trigger. Moreover, coalescence between adjacent compartments within a gel emulsion can be induced using electrical potential. Microfluidics using gel emulsions will be well-suited for combinatorial chemistry, DNA sequencing, drug screening and protein crystallizations.

  13. Microfluidic Mixing Technology for a Universal Health Sensor

    NASA Technical Reports Server (NTRS)

    Chan, Eugene Y.; Bae, Candice

    2009-01-01

    A highly efficient means of microfluidic mixing has been created for use with the rHEALTH sensor an elliptical mixer and passive curvilinear mixing patterns. The rHEALTH sensor provides rapid, handheld, complete blood count, cell differential counts, electrolyte measurements, and other lab tests based on a reusable, flow-based microfluidic platform. These geometries allow for cleaning in a reusable manner, and also allow for complete mixing of fluid streams. The microfluidic mixing is performed by flowing two streams of fluid into an elliptical or curvilinear design that allows the combination of the flows into one channel. The mixing is accomplished by either chaotic advection around micro - fluidic loops. All components of the microfluidic chip are flow-through, meaning that cleaning solution can be introduced into the chip to flush out cells, plasma proteins, and dye. Tests were performed on multiple chip geometries to show that cleaning is efficient in any flowthrough design. The conclusion from these experiments is that the chip can indeed be flushed out with microliter volumes of solution and biological samples are cleaned readily from the chip with minimal effort. The technology can be applied in real-time health monitoring at patient s bedside or in a doctor s office, and real-time clinical intervention in acute situations. It also can be used for daily measurement of hematocrit for patients on anticoagulant drugs, or to detect acute myocardial damage outside a hospital.

  14. Assembly of designed protein scaffolds into monolayers for nanoparticle patterning.

    PubMed

    Mejias, Sara H; Couleaud, Pierre; Casado, Santiago; Granados, Daniel; Garcia, Miguel Angel; Abad, Jose M; Cortajarena, Aitziber L

    2016-05-01

    The controlled assembly of building blocks to achieve new nanostructured materials with defined properties at different length scales through rational design is the basis and future of bottom-up nanofabrication. This work describes the assembly of the idealized protein building block, the consensus tetratricopeptide repeat (CTPR), into monolayers by oriented immobilization of the blocks. The selectivity of thiol-gold interaction for an oriented immobilization has been verified by comparing a non-thiolated protein building block. The physical properties of the CTPR protein thin biomolecular films including topography, thickness, and viscoelasticity, are characterized. Finally, the ability of these scaffolds to act as templates for inorganic nanostructures has been demonstrated by the formation of well-packed gold nanoparticles (GNPs) monolayer patterned by the CTPR monolayer. PMID:26844645

  15. Microfluidic Device

    NASA Technical Reports Server (NTRS)

    Tai, Yu-Chong (Inventor); Zheng, Siyang (Inventor); Lin, Jeffrey Chun-Hui (Inventor); Kasdan, Harvey (Inventor)

    2015-01-01

    Described herein are particular embodiments relating to a microfluidic device that may be utilized for cell sensing, counting, and/or sorting. Particular aspects relate to a microfabricated device that is capable of differentiating single cell types from dense cell populations. One particular embodiment relates a device and methods of using the same for sensing, counting, and/or sorting leukocytes from whole, undiluted blood samples.

  16. Microfluidic Device

    NASA Technical Reports Server (NTRS)

    Tai, Yu-Chong (Inventor); Zheng, Siyang (Inventor); Lin, Jeffrey Chun-Hui (Inventor); Kasdan, Harvey L. (Inventor)

    2016-01-01

    Described herein are particular embodiments relating to a microfluidic device that may be utilized for cell sensing, counting, and/or sorting. Particular aspects relate to a microfabricated device that is capable of differentiating single cell types from dense cell populations. One particular embodiment relates a device and methods of using the same for sensing, counting, and/or sorting leukocytes from whole, undiluted blood samples.

  17. Functional module identification in protein interaction networks by interaction patterns

    PubMed Central

    Wang, Yijie; Qian, Xiaoning

    2014-01-01

    Motivation: Identifying functional modules in protein–protein interaction (PPI) networks may shed light on cellular functional organization and thereafter underlying cellular mechanisms. Many existing module identification algorithms aim to detect densely connected groups of proteins as potential modules. However, based on this simple topological criterion of ‘higher than expected connectivity’, those algorithms may miss biologically meaningful modules of functional significance, in which proteins have similar interaction patterns to other proteins in networks but may not be densely connected to each other. A few blockmodel module identification algorithms have been proposed to address the problem but the lack of global optimum guarantee and the prohibitive computational complexity have been the bottleneck of their applications in real-world large-scale PPI networks. Results: In this article, we propose a novel optimization formulation LCP2 (low two-hop conductance sets) using the concept of Markov random walk on graphs, which enables simultaneous identification of both dense and sparse modules based on protein interaction patterns in given networks through searching for LCP2 by random walk. A spectral approximate algorithm SLCP2 is derived to identify non-overlapping functional modules. Based on a bottom-up greedy strategy, we further extend LCP2 to a new algorithm (greedy algorithm for LCP2) GLCP2 to identify overlapping functional modules. We compare SLCP2 and GLCP2 with a range of state-of-the-art algorithms on synthetic networks and real-world PPI networks. The performance evaluation based on several criteria with respect to protein complex prediction, high level Gene Ontology term prediction and especially sparse module detection, has demonstrated that our algorithms based on searching for LCP2 outperform all other compared algorithms. Availability and implementation: All data and code are available at http://www.cse.usf.edu/∼xqian/fmi/slcp2hop

  18. Microfluidic waves.

    PubMed

    Utz, Marcel; Begley, Matthew R; Haj-Hariri, Hossein

    2011-11-21

    The propagation of pressure waves in fluidic channels with elastic covers is discussed in view of applications to flow control in microfluidic devices. A theory is presented which describes pressure waves in the fluid that are coupled to bending waves in the elastic cover. At low frequencies, the lateral bending of the cover dominates over longitudinal bending, leading to propagating, non-dispersive longitudinal pressure waves in the channel. The theory addresses effects due to both the finite viscosity and compressibility of the fluid. The coupled waves propagate without dispersion, as long as the wave length is larger than the channel width. It is shown that in channels of typical microfluidic dimensions, wave velocities in the range of a few 10 m s(-1) result if the channels are covered by films of a compliant material such as PDMS. The application of this principle to design microfluidic band pass filters based on standing waves is discussed. Characteristic frequencies in the range of a few kHz are readily achieved with quality factors above 30. PMID:21966667

  19. New Host Factors Important for Respiratory Syncytial Virus (RSV) Replication Revealed by a Novel Microfluidics Screen for Interactors of Matrix (M) Protein*

    PubMed Central

    Kipper, Sarit; Hamad, Samar; Caly, Leon; Avrahami, Dorit; Bacharach, Eran; Jans, David A.; Gerber, Doron; Bajorek, Monika

    2015-01-01

    Although human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and elderly worldwide, there is no licensed RSV vaccine or effective drug treatment available. The RSV Matrix protein plays key roles in virus life cycle, being found in the nucleus early in infection in a transcriptional inhibitory role, and later localizing in viral inclusion bodies before coordinating viral assembly and budding at the plasma membrane. In this study, we used a novel, high throughput microfluidics platform and custom human open reading frame library to identify novel host cell binding partners of RSV matrix. Novel interactors identified included proteins involved in host transcription regulation, the innate immunity response, cytoskeletal regulation, membrane remodeling, and cellular trafficking. A number of these interactions were confirmed by immunoprecipitation and cellular colocalization approaches. Importantly, the physiological significance of matrix interaction with the actin-binding protein cofilin 1, caveolae protein Caveolin 2, and the zinc finger protein ZNF502 was confirmed. siRNA knockdown of the host protein levels resulted in reduced RSV virus production in infected cells. These results have important implications for future antiviral strategies aimed at targets of RSV matrix in the host cell. PMID:25556234

  20. Improved method for predicting protein fold patterns with ensemble classifiers.

    PubMed

    Chen, W; Liu, X; Huang, Y; Jiang, Y; Zou, Q; Lin, C

    2012-01-01

    Protein folding is recognized as a critical problem in the field of biophysics in the 21st century. Predicting protein-folding patterns is challenging due to the complex structure of proteins. In an attempt to solve this problem, we employed ensemble classifiers to improve prediction accuracy. In our experiments, 188-dimensional features were extracted based on the composition and physical-chemical property of proteins and 20-dimensional features were selected using a coupled position-specific scoring matrix. Compared with traditional prediction methods, these methods were superior in terms of prediction accuracy. The 188-dimensional feature-based method achieved 71.2% accuracy in five cross-validations. The accuracy rose to 77% when we used a 20-dimensional feature vector. These methods were used on recent data, with 54.2% accuracy. Source codes and dataset, together with web server and software tools for prediction, are available at: http://datamining.xmu.edu.cn/main/~cwc/ProteinPredict.html. PMID:22370884

  1. Pbx Homeodomain Proteins: TALEnted regulators of Limb Patterning and Outgrowth

    PubMed Central

    Capellini, Terence D.; Zappavigna, Vincenzo; Selleri, Licia

    2011-01-01

    Limb development has long provided an excellent model for understanding the genetic principles driving embryogenesis. Studies utilizing chick and mouse have led to new insights into limb patterning and morphogenesis. Recent research has centered on the regulatory networks underlying limb development. Here, we discuss the hierarchical, overlapping, and iterative roles of Pbx family members in appendicular development that have emerged from genetic analyses in the mouse. Pbx genes are essential in determining limb bud positioning, early bud formation, limb axes establishment and coordination, and patterning and morphogenesis of most elements of the limb and girdle. Pbx proteins directly regulate critical effectors of limb and girdle development, including morphogen-encoding genes like Shh in limb posterior mesoderm, and transcription factor-encoding genes like Alx1 in pre-scapular domains. Interestingly, at least in limb buds, Pbx appear to act not only as Hox cofactors, but also in the upstream control of 5' HoxA/D gene expression. PMID:21416555

  2. Tacky COC: a solvent bonding technique for fabrication of microfluidic systems

    NASA Astrophysics Data System (ADS)

    Keller, Nico; Nargang, Tobias M.; Helmer, Dorothea; Rapp, Bastian E.

    2016-03-01

    The academic community knows cyclic olefin copolymer (COC) as a well suited material for microfluidic applications because COC has numerous interesting properties such as high transmittance, good chemical resistance and good biocompatibility. Here we present a fast and cost-effective method for bonding of two COC substrates: exposure to appropriate solvents gives a tacky COC surface which when brought in contact with untreated COC forms a strong and optical clear bond. The bonding process is carried out at room temperature and takes less than three minutes which makes it significantly faster than currently described methods: This method does not require special lab equipment such as hot plates or hydraulic presses. The mild conditions of the bond process also allow for such "tacky COC" lids to be used for sealing of microfluidic chips containing immobilized protein patterns which is of high interest for immunodiagnostic testing inside microfluidic chips.

  3. Concentration Dependent Ion-Protein Interaction Patterns Underlying Protein Oligomerization Behaviours.

    PubMed

    Batoulis, Helena; Schmidt, Thomas H; Weber, Pascal; Schloetel, Jan-Gero; Kandt, Christian; Lang, Thorsten

    2016-01-01

    Salts and proteins comprise two of the basic molecular components of biological materials. Kosmotropic/chaotropic co-solvation and matching ion water affinities explain basic ionic effects on protein aggregation observed in simple solutions. However, it is unclear how these theories apply to proteins in complex biological environments and what the underlying ionic binding patterns are. Using the positive ion Ca(2+) and the negatively charged membrane protein SNAP25, we studied ion effects on protein oligomerization in solution, in native membranes and in molecular dynamics (MD) simulations. We find that concentration-dependent ion-induced protein oligomerization is a fundamental chemico-physical principle applying not only to soluble but also to membrane-anchored proteins in their native environment. Oligomerization is driven by the interaction of Ca(2+) ions with the carboxylate groups of aspartate and glutamate. From low up to middle concentrations, salt bridges between Ca(2+) ions and two or more protein residues lead to increasingly larger oligomers, while at high concentrations oligomers disperse due to overcharging effects. The insights provide a conceptual framework at the interface of physics, chemistry and biology to explain binding of ions to charged protein surfaces on an atomistic scale, as occurring during protein solubilisation, aggregation and oligomerization both in simple solutions and membrane systems. PMID:27052788

  4. Concentration Dependent Ion-Protein Interaction Patterns Underlying Protein Oligomerization Behaviours

    PubMed Central

    Batoulis, Helena; Schmidt, Thomas H.; Weber, Pascal; Schloetel, Jan-Gero; Kandt, Christian; Lang, Thorsten

    2016-01-01

    Salts and proteins comprise two of the basic molecular components of biological materials. Kosmotropic/chaotropic co-solvation and matching ion water affinities explain basic ionic effects on protein aggregation observed in simple solutions. However, it is unclear how these theories apply to proteins in complex biological environments and what the underlying ionic binding patterns are. Using the positive ion Ca2+ and the negatively charged membrane protein SNAP25, we studied ion effects on protein oligomerization in solution, in native membranes and in molecular dynamics (MD) simulations. We find that concentration-dependent ion-induced protein oligomerization is a fundamental chemico-physical principle applying not only to soluble but also to membrane-anchored proteins in their native environment. Oligomerization is driven by the interaction of Ca2+ ions with the carboxylate groups of aspartate and glutamate. From low up to middle concentrations, salt bridges between Ca2+ ions and two or more protein residues lead to increasingly larger oligomers, while at high concentrations oligomers disperse due to overcharging effects. The insights provide a conceptual framework at the interface of physics, chemistry and biology to explain binding of ions to charged protein surfaces on an atomistic scale, as occurring during protein solubilisation, aggregation and oligomerization both in simple solutions and membrane systems. PMID:27052788

  5. Fragile X mental retardation protein (FMRP) interacting proteins exhibit different expression patterns during development.

    PubMed

    Bonaccorso, C M; Spatuzza, M; Di Marco, B; Gloria, A; Barrancotto, G; Cupo, A; Musumeci, S A; D'Antoni, S; Bardoni, B; Catania, M V

    2015-05-01

    Fragile X syndrome is caused by the lack of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA transport and translation. FMRP is a component of mRNA ribonucleoprotein complexes and it can interact with a range of proteins either directly or indirectly, as demonstrated by two-hybrid selection and co-immunoprecipitation, respectively. Most of FMRP-interacting proteins are RNA-binding proteins such as FXR1P, FXR2P and 82-FIP. Interestingly, FMRP can also interact directly with the cytoplasmic proteins CYFIP1 and CYFIP2, which do not bind RNA and link FMRP to the RhoGTPase pathway. The interaction with these different proteins may modulate the functions of FMRP by influencing its affinity to RNA and by affecting the FMRP ability of cytoskeleton remodeling through Rho/Rac GTPases. To better define the relationship of FMRP with its interacting proteins during brain development, we have analyzed the expression pattern of FMRP and its interacting proteins in the cortex, striatum, hippocampus and cerebellum at different ages in wild type (WT) mice. FMRP and FXR2P were strongly expressed during the first week and gradually decreased thereafter, more rapidly in the cerebellum than in the cortex. FXR1P was also expressed early and showed a reduction at later stages of development with a similar developmental pattern in these two regions. CYFIP1 was expressed at all ages and peaked in the third post-natal week. In contrast, CYFIP2 and 82-FIP (only in forebrain regions) were moderately expressed at P3 and gradually increased after P7. In general, the expression pattern of each protein was similar in the regions examined, except for 82-FIP, which exhibited a strong expression at P3 and low levels at later developmental stages in the cerebellum. Our data indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in

  6. The E4 protein; structure, function and patterns of expression

    SciTech Connect

    Doorbar, John

    2013-10-15

    }E4, these kinases regulate one of the E1{sup ∧}E4 proteins main functions, the association with the cellular keratin network, and eventually also its cleavage by the protease calpain which allows assembly into amyloid-like fibres and reorganisation of the keratin network. Although the E4 proteins of different HPV types appear divergent at the level of their primary amino acid sequence, they share a recognisable modular organisation and pattern of expression, which may underlie conserved functions and regulation. Assembly into higher-order multimers and suppression of cell proliferation are common to all E4 proteins examined. Although not yet formally demonstrated, a role in virus release and transmission remains a likely function for E4. - Highlights: • E4 gene products have a modular structure, and are expressed from the E1{sup ∧}E4 spliced mRNA. • E4 proteins are modified during epithelial differentiation by phosphorylation and proteolysis. • The E4 proteins contribute to genome amplification-efficiency and virus synthesis. • E4 proteins are abundantly expressed and may facilitate efficient virus release and transmission. • High-risk E4 proteins are deposited as amyloid fibres and can be used as infection biomarkers.

  7. Pattern Discovery in Breast Cancer Specific Protein Interaction Network

    PubMed Central

    Wu, Xiaogang; Harrison, Scott H.; Chen, Jake Yue

    2009-01-01

    The interest in indentifying novel biomarkers for early stage breast cancer (BRCA) detection has become grown significantly in recent years. From a view of network biology, one of the emerging themes today is to re-characterize a protein’s biological functions in its molecular network. Although many methods have been presented, including network-based gene ranking for molecular biomarker discovery, and graph clustering for functional module discovery, it is still hard to find systems-level properties hidden in disease specific molecular networks. We reconstructed BRCA-related protein interaction network by using BRCA-associated genes/proteins as seeds, and expanding them in an integrated protein interaction database. We further developed a computational framework based on Ant Colony Optimization to rank network nodes. The task of ranking nodes is represented as the problem of finding optimal density distributions of “ant colonies” on all nodes of the network. Our results revealed some interesting systems-level pattern in BRCA-related protein interaction network. PMID:21347162

  8. Microfluidic Channels on Nanopatterned Substrates: Monitoring Protein Binding to Lipid Bilayers with Surface-Enhanced Raman Spectroscopy

    PubMed Central

    Banerjee, Amrita; Perez-Castillejos, R.; Hahn, D.; Smirnov, Alex I.; Grebel, H.

    2013-01-01

    We used Surface Enhanced Raman Spectroscopy (SERS) to detect binding events between streptavidin and biotinylated lipid bilayers. The binding events took place at the surface between microfluidic channels and anodized aluminum oxide (AAO) with the latter serving as substrates. The bilayers were incorporated in the substrate pores. It was revealed that non-bound molecules were easily washed away and that large suspended cells (Salmonella enterica) are less likely to interfere with the monitoring process: when focusing to the lower surface of the channel, one may resolve mostly the bound molecules. PMID:24932024

  9. Microfluidic device having an immobilized pH gradient and PAGE gels for protein separation and analysis

    DOEpatents

    Sommer, Gregory J.; Hatch, Anson V.; Singh, Anup K.; Wang, Ying-Chih

    2012-12-11

    Disclosed is a novel microfluidic device enabling on-chip implementation of a two-dimensional separation methodology. Previously disclosed microscale immobilized pH gradients (IPG) are combined with perpendicular polyacrylamide gel electrophoresis (PAGE) microchannels to achieve orthogonal separations of biological samples. Device modifications enable inclusion of sodium dodecyl sulfate (SDS) in the second dimension. The device can be fabricated to use either continuous IPG gels, or the microscale isoelectric fractionation membranes we have also previously disclosed, for the first dimension. The invention represents the first all-gel two-dimensional separation microdevice, with significantly higher resolution power over existing devices.

  10. Microfluidic device having an immobilized pH gradient and page gels for protein separation and analysis

    DOEpatents

    Sommer, Gregory J; Hatch, Anson V; Singh, Anup K; Wang, Ying-Chih

    2014-05-20

    Disclosed is a novel microfluidic device enabling on-chip implementation of a two-dimensional separation methodology. Previously disclosed microscale immobilized pH gradients (IPG) are combined with perpendicular polyacrylamide gel electrophoresis (PAGE) microchannels to achieve orthogonal separations of biological samples. Device modifications enable inclusion of sodium dodecyl sulfate (SDS) in the second dimension. The device can be fabricated to use either continuous IPG gels, or the microscale isoelectric fractionation membranes we have also previously disclosed, for the first dimension. The invention represents the first all-gel two-dimensional separation microdevice, with significantly higher resolution power over existing devices.

  11. Microfluidic interconnects

    DOEpatents

    Benett, William J.; Krulevitch, Peter A.

    2001-01-01

    A miniature connector for introducing microliter quantities of solutions into microfabricated fluidic devices. The fluidic connector, for example, joins standard high pressure liquid chromatography (HPLC) tubing to 1 mm diameter holes in silicon or glass, enabling ml-sized volumes of sample solutions to be merged with .mu.l-sized devices. The connector has many features, including ease of connect and disconnect; a small footprint which enables numerous connectors to be located in a small area; low dead volume; helium leak-tight; and tubing does not twist during connection. Thus the connector enables easy and effective change of microfluidic devices and introduction of different solutions in the devices.

  12. Laser Ablation of Polymer Microfluidic Devices

    NASA Astrophysics Data System (ADS)

    Killeen, Kevin

    2004-03-01

    Microfluidic technology is ideal for processing precious samples of limited volumes. Some of the most important classes of biological samples are both high in sample complexity and low in concentration. Combining the elements of sample pre-concentration, chemical separation and high sensitivity detection with chemical identification is essential for realizing a functional microfluidic based analysis system. Direct write UV laser ablation has been used to rapidly fabricate microfluidic devices capable of high performance liquid chromatography (HPLC)-MS. These chip-LC/MS devices use bio-compatible, solvent resistant and flexible polymer materials such as polyimide. A novel microfluidic to rotary valve interface enables, leak free, high pressure fluid switching between multiple ports of the microfluidic chip-LC/MS device. Electrospray tips with outer dimension of 50 um and inner of 15 um are formed by ablating the polymer material concentrically around a multilayer laminated channel structure. Biological samples of digested proteins were used to evaluate the performance of these microfluidic devices. Liquid chromatography separation and similar sample pretreatments have been performed using polymeric microfluidic devices with on-chip separation channels. Mass spectrometry was performed using an Agilent Technologies 1100 series ion trap mass spectrometer. Low fmol amounts of protein samples were positively and routinely identified by searching the MS/MS spectral data against protein databases. The sensitivity and separation performance of the chip-LC devices has been found to be comparable to state of the art nano-electrospray systems.

  13. Preparation of stripe-patterned heterogeneous hydrogel sheets using microfluidic devices for high-density coculture of hepatocytes and fibroblasts.

    PubMed

    Kobayashi, Aoi; Yamakoshi, Kenta; Yajima, Yuya; Utoh, Rie; Yamada, Masumi; Seki, Minoru

    2013-12-01

    Here we demonstrate the production of stripe-patterned heterogeneous hydrogel sheets for the high-density 3D coculture of multiple cell types, by using microchannel-combined micronozzle devices. The prepared hydrogel sheet, composed of multiple regions with varying physical stiffness, regulates the direction of proliferation of encapsulated cells and enables the formation of arrays of rod-like heterotypic organoids inside the hydrogel matrix. We successfully prepared stripe-patterned hydrogel sheets with a uniform thickness of ~100 μm and a width of several millimeters. Hepatoma cells (HepG2) and fibroblasts (Swiss 3T3) were embedded inside the hydrogel matrix and cocultured, to form heterotypic micro-organoids mimicking in vivo hepatic cord structures. The upregulation of hepatic functions by the 3D coculture was confirmed by analyzing liver-specific functions. The presented heterogeneous hydrogel sheet could be useful, as it provides relatively large, but precisely-controlled, 3-dimensional microenvironments for the high-density coculture of multiple types of cells. PMID:23845912

  14. Using macromolecular-crystallography beamline and microfluidic platform for small-angle diffraction studies of lipidic matrices for membrane-protein crystallization

    NASA Astrophysics Data System (ADS)

    Kondrashkina, E.; Khvostichenko, D. S.; Perry, S. L.; Von Osinski, J.; Kenis, P. J. A.; Brister, K.

    2013-03-01

    Macromolecular-crystallography (MX) beamlines routinely provide a possibility to change X-ray beam energy, focus the beam to a size of tens of microns, align a sample on a microdiffractometer using on-axis video microscope, and collect data with an area-detector positioned in three dimensions. These capabilities allow for running complementary measurements of small-angle X-ray scattering and diffraction (SAXS) at the same beamline with such additions to the standard MX setup as a vacuum path between the sample and the detector, a modified beam stop, and a custom sample cell. On the 21-ID-D MX beamline at the Advanced Photon Source we attach a vacuum flight tube to the area detector support and use the support motion for aligning a beam stop built into the rear end of the flight tube. At 8 KeV energy and 1 m sample-to-detector distance we can achieve a small-angle resolution of 0.01A-1 in the reciprocal space. Measuring SAXS with this setup, we have studied phase diagrams of lipidic mesophases used as matrices for membrane-protein crystallization. The outcome of crystallization trials is significantly affected by the structure of the lipidic mesophases, which is determined by the composition of the crystallization mixture. We use a microfluidic chip for the mesophase formulation and in situ SAXS data collection. Using the MX beamline and the microfluidic platform we have demonstrated the viability of the high-throughput SAXS studies facilitating screening of lipidic matrices for membrane-protein crystallization.

  15. Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution

    SciTech Connect

    Wu, Meiye; Singh, Anup K.

    2014-07-15

    In this study, cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation kinase cascade that culminates in induction of mRNA and non-coding miRNA production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient post-translational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR for nucleic acids, and flow cytometry for post-translational modifications. Since we know that cells in populations behave heterogeneously1, especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell’s physiological state. In this technical brief, we describe our microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and post-translational modifications in single intact cells with >95% reduction in reagent requirement in under 8 hours.

  16. Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution

    DOE PAGESBeta

    Wu, Meiye; Singh, Anup K.

    2014-07-15

    In this study, cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation kinase cascade that culminates in induction of mRNA and non-coding miRNA production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient post-translational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR formore » nucleic acids, and flow cytometry for post-translational modifications. Since we know that cells in populations behave heterogeneously1, especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell’s physiological state. In this technical brief, we describe our microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and post-translational modifications in single intact cells with >95% reduction in reagent requirement in under 8 hours.« less

  17. Altered glycosylation pattern of proteins in Alzheimer disease.

    PubMed

    Guevara, J; Espinosa, B; Zenteno, E; Vázguez, L; Luna, J; Perry, G; Mena, R

    1998-10-01

    Post-translational modifications due to glycosylation of proteins in human brains from patients with Alzheimer disease (AD) were analyzed using lectin histochemistry. Results indicate a significant increase in the production of O-glycosylated (containing Galbeta1,3GalNAc alpha1,0 Ser/Thr or GalNAc alpha1,0 Ser/Thr) proteins in neuritic plaques and neurofibrillary tangles which are the major histopathological hallmarks of AD brains. These alterations were determined by positive labelling with lectins obtained from Amaranthus leucocarpus (ALL) and Macrobrachium rosenbergii (MRL) respectively. Immunohistochemistry indicated that the lectin-staining labelled specifically both neurofibrillary tangles and neuritic plaques. In contrast, lectins labelling was restricted to microvessels in normal control brains. These results provide evidence that modifications of the specific glycosylation patterns are closely related with the presence of the hallmark lesions of this disease, suggesting that an abnormal enzymatic processing of proteins may be an early event in the neuronal degeneration which characterises AD. PMID:9786241

  18. Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

    PubMed Central

    Hou, Yanxia; Genua, Maria; Garçon, Laurie-Amandine; Buhot, Arnaud; Calemczuk, Roberto; Bonnaffé, David; Lortat-Jacob, Hugues; Livache, Thierry

    2014-01-01

    In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins. PMID:25286325

  19. Electronic tongue generating continuous recognition patterns for protein analysis.

    PubMed

    Hou, Yanxia; Genua, Maria; Garçon, Laurie-Amandine; Buhot, Arnaud; Calemczuk, Roberto; Bonnaffé, David; Lortat-Jacob, Hugues; Livache, Thierry

    2014-01-01

    In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins. PMID:25286325

  20. Semiconductor sensor embedded microfluidic chip for protein biomarker detection using a bead-based immunoassay combined with deoxyribonucleic acid strand labeling.

    PubMed

    Lin, Yen-Heng; Peng, Po-Yu

    2015-04-15

    Two major issues need to be addressed in applying semiconductor biosensors to detecting proteins in immunoassays. First, the length of the antibody on the sensor surface surpasses the Debye lengths (approximately 1 nm, in normal ionic strength solution), preventing certain specifically bound proteins from being tightly attached to the sensor surface. Therefore, these proteins do not contribute to the sensor's surface potential change. Second, these proteins carry a small charge and can be easily affected by the pH of the surrounding solution. This study proposes a magnetic bead-based immunoassay using a secondary antibody to label negatively charged DNA fragments for signal amplification. An externally imposed magnetic force attaches the analyte tightly to the sensor surface, thereby effectively solving the problem of the analyte protein's distance to the sensor surface surpassing the Debye lengths. In addition, a normal ion intensity buffer can be used without dilution for the proposed method. Experiments revealed that the sensitivity can be improved by using a longer DNA fragment for labeling and smaller magnetic beads as solid support for the antibody. By using a 90 base pair DNA label, the signal was 15 times greater than that without labeling. In addition, by using a 120 nm magnetic bead, a minimum detection limit of 12.5 ng mL(-1) apolipoprotein A1 can be measured. Furthermore, this study integrates a semiconductor sensor with a microfluidic chip. With the help of microvalves and micromixers in the chip, the length of the mixing step for each immunoassay has been reduced from 1h to 20 min, and the sample volume has been reduced from 80 μL to 10 μL. In practice, a protein biomarker in a urinary bladder cancer patient's urine was successfully measured using this technique. This study provides a convenient and effective method to measure protein using a semiconductor sensor. PMID:25818137

  1. Microfluidic electrochemical reactors

    DOEpatents

    Nuzzo, Ralph G.; Mitrovski, Svetlana M.

    2011-03-22

    A microfluidic electrochemical reactor includes an electrode and one or more microfluidic channels on the electrode, where the microfluidic channels are covered with a membrane containing a gas permeable polymer. The distance between the electrode and the membrane is less than 500 micrometers. The microfluidic electrochemical reactor can provide for increased reaction rates in electrochemical reactions using a gaseous reactant, as compared to conventional electrochemical cells. Microfluidic electrochemical reactors can be incorporated into devices for applications such as fuel cells, electrochemical analysis, microfluidic actuation, pH gradient formation.

  2. Hydrodynamic guiding for addressing subsets of immobilized cells and molecules in microfluidic systems

    PubMed Central

    Brevig, Thomas; Krühne, Ulrich; Kahn, Rachel A; Ahl, Thomas; Beyer, Michael; Pedersen, Lars H

    2003-01-01

    Background The interest in microfluidics and surface patterning is increasing as the use of these technologies in diverse biomedical applications is substantiated. Controlled molecular and cellular surface patterning is a costly and time-consuming process. Methods for keeping multiple separate experimental conditions on a patterned area are, therefore, needed to amplify the amount of biological information that can be retrieved from a patterned surface area. We describe, in three examples of biomedical applications, how this can be achieved in an open microfluidic system, by hydrodynamically guiding sample fluid over biological molecules and living cells immobilized on a surface. Results A microfluidic format of a standard assay for cell-membrane integrity showed a fast and dose-dependent toxicity of saponin on mammalian cells. A model of the interactions of human mononuclear leukocytes and endothelial cells was established. By contrast to static adhesion assays, cell-cell adhesion in this dynamic model depended on cytokine-mediated activation of both endothelial and blood cells. The microfluidic system allowed the use of unprocessed blood as sample material, and a specific and fast immunoassay for measuring the concentration of C-reactive protein in whole blood was demonstrated. Conclusion The use of hydrodynamic guiding made multiple and dynamic experimental conditions on a small surface area possible. The ability to change the direction of flow and produce two-dimensional grids can increase the number of reactions per surface area even further. The described microfluidic system is widely applicable, and can take advantage of surfaces produced by current and future techniques for patterning in the micro- and nanometer scale. PMID:12875662

  3. Microfluidic interconnects

    DOEpatents

    Benett, William J.; Krulevitch, Peter A.

    2001-01-01

    A miniature connector for introducing microliter quantities of solutions into microfabricated fluidic devices, and which incorporates a molded ring or seal set into a ferrule cartridge, with or without a compression screw. The fluidic connector, for example, joins standard high pressure liquid chromatography (HPLC) tubing to 1 mm diameter holes in silicon or glass, enabling ml-sized volumes of sample solutions to be merged with .mu.l-sized devices. The connector has many features, including ease of connect and disconnect; a small footprint which enables numerous connectors to be located in a small area; low dead volume; helium leak-tight; and tubing does not twist during connection. Thus the connector enables easy and effective change of microfluidic devices and introduction of different solutions in the devices.

  4. Fabrication of microfluidic vascular phantoms by laser micromachining

    NASA Astrophysics Data System (ADS)

    Mathews, Scott A.; Luu, Long; Ramella-Roman, Jessica C.

    2012-06-01

    Imaging of capillary structures and monitoring of blood flow within vasculature is becoming more common in clinical settings. However, very few dynamic phantoms exist which mimic capillary structures. We report the fabrication and testing of microfluidic, vascular phantoms aimed at the study of blood flow. These phantoms are fabricated using low-cost, off-the-shelf materials and require no lithographic processing, stamping, or embossing. Using laser micromachining, complex microfluidic structures can be fabricated in under an hour. The laser system is capable of producing microfluidic features with sizes on the order of tens of microns, over an area of several square centimeters. Because the laser micromachining system is computer controlled and accepts both vector and raster files, the microfluidic structure can be simple, rectilinear patterns or complex, anatomically correct patterns. The microfluidic devices interface with simple off the shelf syringe pumps. The microfluidic devices fabricated with this technique were used for non-invasive monitoring of flow using speckle based techniques.

  5. Expression pattern of Protein Kinase C ϵ during mouse embryogenesis

    PubMed Central

    2013-01-01

    Background Protein kinase C epsilon (PKCϵ) belongs to the novel PKC subfamily, which consists of diacylglycerol dependent- and calcium independent-PKCs. Previous studies have shown that PKCϵ is important in different contexts, such as wound healing or cancer. In this study, we contribute to expand the knowledge on PKCϵ by reporting its expression pattern during murine midgestation using the LacZ reporter gene and immunostaining procedures. Results Sites showing highest PKCϵ expression were heart at ealier stages, and ganglia in older embryos. Other stained domains included somites, bone, stomach, kidney, and blood vessels. Conclusions The seemingly strong expression of PKCϵ in heart and ganglia shown in this study suggests a important role of this isoform in the vascular and nervous systems during mouse development. However, functional redundancy with other PKCs during midgestation within these domains and others reported here possibly exists since PKCϵ deficient mice do not display obvious embryonic developmental defects. PMID:23639204

  6. Protein patterning by microcontact printing using pyramidal PDMS stamps.

    PubMed

    Filipponi, Luisa; Livingston, Peter; Kašpar, Ondřej; Tokárová, Viola; Nicolau, Dan V

    2016-02-01

    Micro-contact printing, μCP, is a well-established soft-lithography technique for printing biomolecules. μCP uses stamps made of Poly(dimethylsiloxane), PDMS, made by replicating a microstructured silicon master fabricated by semiconductor manufacturing processes. One of the problems of the μCP is the difficult control of the printing process, which, because of the high compressibility of PDMS, is very sensitive to minute changes in the applied pressure. This over-sensitive response leads to frequent and/or uncontrollable collapse of the stamps with high aspect ratios, thus decreasing the printing accuracy and reproducibility. Here we present a straightforward methodology of designing and fabricating PDMS structures with an architecture which uses the collapse of the stamp to reduce, rather than enlarge the variability of the printing. The PDMS stamp, organized as an array of pyramidal micro-posts, whose ceiling collapses when pressed on a flat surface, replicates the structure of the silicon master fabricated by anisotropic wet etching. Upon application of pressure, depending on the size of, and the pitch between, the PDMS pyramids, an air gap is formed surrounding either the entire array, or individual posts. The printing technology, which also exhibits a remarkably low background noise for fluorescence detection, may find applications when the clear demarcation of the shapes of protein patterns and the distance between them are critical, such as microarrays and studies of cell patterning. PMID:26782964

  7. Tacky cyclic olefin copolymer: a biocompatible bonding technique for the fabrication of microfluidic channels in COC.

    PubMed

    Keller, Nico; Nargang, Tobias M; Runck, Matthias; Kotz, Frederik; Striegel, Andreas; Sachsenheimer, Kai; Klemm, Denis; Länge, Kerstin; Worgull, Matthias; Richter, Christiane; Helmer, Dorothea; Rapp, Bastian E

    2016-04-26

    Cyclic olefin copolymer (COC) is widely used in microfluidics due to its UV-transparency, its biocompatibility and high chemical resistance. Here we present a fast and cost-effective solvent bonding technique, which allows for the efficient bonding of protein-patterned COC structures. The bonding process is carried out at room temperature and takes less than three minutes. Enzyme activity is retained upon bonding and microstructure deformation does not occur. PMID:27040493

  8. Superhydrophobicity for antifouling microfluidic surfaces.

    PubMed

    Shirtcliffe, N J; Roach, P

    2013-01-01

    Fouling of surfaces is often problematic in microfluidic devices, particularly when using protein or -enzymatic solutions. Various coating methods have been investigated to reduce the tendency for protein molecules to adsorb, mostly relying on hydrophobic surface chemistry or the antifouling ability of -polyethylene glycol. Here we present the potential use of superhydrophobic surfaces to not only reduce the amount of surface contamination but also to induce self-cleaning under flow conditions. The methodology is presented in order to prepare superhydrophobic surface coatings having micro- and nanoscale feature dimensions, as well as a step-by-step guide to quantify adsorbed protein down to nanogram levels. The fabrication of these surfaces as coatings via silica sol-gel and copper nano-hair growth is presented, which can be applied within microfluidic devices manufactured from various materials. PMID:23329449

  9. Microfluidic System for Solution Array Based Bioassays

    SciTech Connect

    Dougherty, G M; Tok, J B; Pannu, S S; Rose, K A

    2006-02-10

    The objective of this project is to demonstrate new enabling technology for multiplex biodetection systems that are flexible, miniaturizable, highly automated, low cost, and high performance. It builds on prior successes at LLNL with particle-based solution arrays, such as those used in the Autonomous Pathogen Detection System (APDS) successfully field deployed to multiple locations nationwide. We report the development of a multiplex solution array immunoassay based upon engineered metallic nanorod particles. Nanobarcodes{reg_sign} particles are fabricated by sequential electrodeposition of dissimilar metals within porous alumina templates, yielding optically encoded striping patterns that can be read using standard laboratory microscope optics and PC-based image processing software. The addition of self-assembled monolayer (SAM) coatings and target-specific antibodies allows each encoded class of nanorod particles to be directed against a different antigen target. A prototype assay panel directed against bacterial, viral, and soluble protein targets demonstrates simultaneous detection at sensitivities comparable to state of the art immunoassays, with minimal cross-reactivity. Studies have been performed to characterize the colloidal properties (zeta potential) of the suspended nanorod particles as a function of pH, the ionic strength of the suspending solution, and surface functionalization state. Additional studies have produced means for the non-contact manipulation of the particles, including the insertion of magnetic nickel stripes within the encoding pattern, and control via externally applied electromagnetic fields. Using the results of these studies, the novel Nanobarcodes{reg_sign} based assay was implemented in a prototype automated system with the sample processing functions and optical readout performed on a microfluidic card. The unique physical properties of the nanorod particles enable the development of integrated microfluidic systems for

  10. PREFACE: Nano- and microfluidics Nano- and microfluidics

    NASA Astrophysics Data System (ADS)

    Jacobs, Karin

    2011-05-01

    The field of nano- and microfluidics emerged at the end of the 1990s parallel to the demand for smaller and smaller containers and channels for chemical, biochemical and medical applications such as blood and DNS analysis [1], gene sequencing or proteomics [2, 3]. Since then, new journals and conferences have been launched and meanwhile, about two decades later, a variety of microfluidic applications are on the market. Briefly, 'the small flow becomes mainstream' [4]. Nevertheless, research in nano- and microfluidics is more than downsizing the spatial dimensions. For liquids on the nanoscale, surface and interface phenomena grow in importance and may even dominate the behavior in some systems. The studies collected in this special issue all concentrate on these type of systems and were part ot the priority programme SPP1164 'Nano- and Microfluidics' of the German Science Foundation (Deutsche Forschungsgemeinschaft, DFG). The priority programme was initiated in 2002 by Hendrik Kuhlmann and myself and was launched in 2004. Friction between a moving liquid and a solid wall may, for instance, play an important role so that the usual assumption of a no-slip boundary condition is no longer valid. Likewise, the dynamic deformations of soft objects like polymers, vesicles or capsules in flow arise from the subtle interplay between the (visco-)elasticity of the object and the viscous stresses in the surrounding fluid and, potentially, the presence of structures confining the flow like channels. Consequently, new theories were developed ( see articles in this issue by Münch and Wagner, Falk and Mecke, Bonthuis et al, Finken et al, Almenar and Rauscher, Straube) and experiments were set up to unambiguously demonstrate deviations from bulk, or 'macro', behavior (see articles in this issue by Wolff et al, Vinogradova and Belyaev, Hahn et al, Seemann et al, Grüner and Huber, Müller-Buschbaum et al, Gutsche et al, Braunmüller et al, Laube et al, Brücker, Nottebrock et al

  11. Microfluidic sieve valves

    SciTech Connect

    Quake, Stephen R; Marcus, Joshua S; Hansen, Carl L

    2015-01-13

    Sieve valves for use in microfluidic device are provided. The valves are useful for impeding the flow of particles, such as chromatography beads or cells, in a microfluidic channel while allowing liquid solution to pass through the valve. The valves find particular use in making microfluidic chromatography modules.

  12. Toward one-step point-of-care immunodiagnostics using capillary-driven microfluidics and PDMS substrates.

    PubMed

    Gervais, Luc; Delamarche, Emmanuel

    2009-12-01

    Point-of-care diagnostics will strongly benefit from miniaturization based on microfluidics because microfluidics integrate functions that can together preserve valuable samples and reagents, increase sensitivity of a test, and accelerate mass transport limited reactions. But a main challenge is to incorporate reagents into microfluidics and to make microfluidics simple to use. Here, we integrate microfluidic functional elements, some of which were developed earlier, and reagents such as detection antibodies (dAbs), capture antibodies (cAbs) and analyte molecules for making one-step immunoassays: the integrated device only requires the addition of sample to trigger a cascade of events powered by capillary forces for effecting a sandwich immunoassay that is read using a fluorescence microscope. The microfluidic elements comprise a sample collector, delay valves, flow resistors, a deposition zone for dAbs, a reaction chamber sealed with a polydimethylsiloxane (PDMS) substrate, and a capillary pump and vents. Parameters for depositing 3.6 nL of a solution of dAb on the chip using an inkjet are optimized and the PDMS substrate is patterned with analytes, which provide a positive control, and cAbs. Various storage conditions of the patterned PDMS are investigated for up to 6 months revealing that storage with a desiccant preserved at least 51% of the activity of the cAbs. C-reactive protein (CRP), a general inflammation and cardiac marker, is detected using this one-step chip using only 5 microL of human serum by measuring fluorescent signals from 30 x 100 microm(2) areas of the PDMS substrate in the wet reaction chamber. The one-step chip can detect CRP at a concentration of 10 ng mL(-1) in less than 3 min and below 1 ng mL(-1) within 14 min. The work presented here may spur the adoption of fluorescence immunoassays using capillary driven microfluidics and PDMS substrates for point-of-care diagnostics. PMID:19904397

  13. Electro-Microfluidic Packaging

    NASA Astrophysics Data System (ADS)

    Benavides, G. L.; Galambos, P. C.

    2002-06-01

    There are many examples of electro-microfluidic products that require cost effective packaging solutions. Industry has responded to a demand for products such as drop ejectors, chemical sensors, and biological sensors. Drop ejectors have consumer applications such as ink jet printing and scientific applications such as patterning self-assembled monolayers or ejecting picoliters of expensive analytes/reagents for chemical analysis. Drop ejectors can be used to perform chemical analysis, combinatorial chemistry, drug manufacture, drug discovery, drug delivery, and DNA sequencing. Chemical and biological micro-sensors can sniff the ambient environment for traces of dangerous materials such as explosives, toxins, or pathogens. Other biological sensors can be used to improve world health by providing timely diagnostics and applying corrective measures to the human body. Electro-microfluidic packaging can easily represent over fifty percent of the product cost and, as with Integrated Circuits (IC), the industry should evolve to standard packaging solutions. Standard packaging schemes will minimize cost and bring products to market sooner.

  14. Sputtered coatings for microfluidic applications

    SciTech Connect

    Matson, Dean W.; Martin, Peter M.; Bennett, Wendy D.; Johnston, John W.; Stewart, Donald C.; Bonham, Charles C.

    2000-07-01

    Magnetron sputter-deposited features and coatings are finding a broad range of uses in microfluidic devices being developed at the Pacific Northwest National Laboratory. Such features are routinely incorporated into multilayer laminated microfluidic components where specific functionality is required, and where other methods for producing these features have been deemed unacceptable. Applications include electrochemical sensors, heaters and temperature probes, electrical leads and insulation layers, piezoelectric actuators and transducers, and chemical modification of surfaces. Small features, such as those required for the production of microsensor electrodes or miniature resistive heaters on microfluidic chips, were patterned using standard lithographic methods, or with masks produced by laser micromachining processes. Thin-film piezoelectric materials such as aluminum nitride have been deposited at low temperatures for use with temperature sensitive materials. Use of the coating technology and its application in the fabrication of specific microfluidic devices, including a groundwater sensor, miniature piezoelectric ultrasonic transducers and actuators, a polymerase chain reaction thermal cycler, and a microchannel flow diagnostic device, are discussed. Technical issues associated with these coatings, such as adhesion, chemical resistance, and surface defects are also addressed. (c) 2000 American Vacuum Society.

  15. Model for evaluating patterned charge regulation contribution to electrostatic interactions between proteins

    NASA Astrophysics Data System (ADS)

    Hollenbeck, Dawn; Martini, K. Michael; Langner, Andreas; Ross, David; Harkin, Anthony; Nelson, Edward; Thurston, George

    2010-03-01

    We study the pattern-specific work of charging for two spherical model proteins in close proximity in ionic solution, using a grand-canonical partition function together with a coarse-grained, linear Debye-Huckel model to calculate the needed work of charging for each possible proton occupancy configuration. We seek to delineate a parameter-space phase diagram to characterize the circumstances under which patterned charge regulation, attractions due to heterogeneous protein charging patterns, and screened net protein charge could individually dominate the electrostatic portion of the interaction between model particles. Within the model, we place titratable residues in accordance with the tertiary protein structure, as is done in the case of a single protein within the Tanford-Kirkwood protein electrostatics model. We use Monte-Carlo simulation and analytical work to evaluate how the local statistics of the charging patterns on each protein respond to close proximity and relative orientation of neighboring proteins.

  16. Ultrafast microfluidics using surface acoustic waves

    PubMed Central

    Yeo, Leslie Y.; Friend, James R.

    2009-01-01

    We demonstrate that surface acoustic waves (SAWs), nanometer amplitude Rayleigh waves driven at megahertz order frequencies propagating on the surface of a piezoelectric substrate, offer a powerful method for driving a host of extremely fast microfluidic actuation and micro∕bioparticle manipulation schemes. We show that sessile drops can be translated rapidly on planar substrates or fluid can be pumped through microchannels at 1–10 cm∕s velocities, which are typically one to two orders quicker than that afforded by current microfluidic technologies. Through symmetry-breaking, azimuthal recirculation can be induced within the drop to drive strong inertial microcentrifugation for micromixing and particle concentration or separation. Similar micromixing strategies can be induced in the same microchannel in which fluid is pumped with the SAW by merely changing the SAW frequency to rapidly switch the uniform through-flow into a chaotic oscillatory flow by exploiting superpositioning of the irradiated sound waves from the sidewalls of the microchannel. If the flow is sufficiently quiescent, the nodes of the transverse standing wave that arises across the microchannel also allow for particle aggregation, and hence, sorting on nodal lines. In addition, the SAW also facilitates other microfluidic capabilities. For example, capillary waves excited at the free surface of a sessile drop by the SAW underneath it can be exploited for micro∕nanoparticle collection and sorting at nodal points or lines at low powers. At higher powers, the large accelerations off the substrate surface as the SAW propagates across drives rapid destabilization of the drop free surface giving rise to inertial liquid jets that persist over 1–2 cm in length or atomization of the entire drop to produce 1–10 μm monodispersed aerosol droplets, which can be exploited for ink-jet printing, mass spectrometry interfacing, or pulmonary drug delivery. The atomization of polymer∕protein solutions

  17. Microfluidic White Organic Light-Emitting Diode Based on Integrated Patterns of Greenish-Blue and Yellow Solvent-Free Liquid Emitters

    NASA Astrophysics Data System (ADS)

    Kobayashi, Naofumi; Kasahara, Takashi; Edura, Tomohiko; Oshima, Juro; Ishimatsu, Ryoichi; Tsuwaki, Miho; Imato, Toshihiko; Shoji, Shuichi; Mizuno, Jun

    2015-10-01

    We demonstrated a novel microfluidic white organic light-emitting diode (microfluidic WOLED) based on integrated sub-100-μm-wide microchannels. Single-μm-thick SU-8-based microchannels, which were sandwiched between indium tin oxide (ITO) anode and cathode pairs, were fabricated by photolithography and heterogeneous bonding technologies. 1-Pyrenebutyric acid 2-ethylhexyl ester (PLQ) was used as a solvent-free greenish-blue liquid emitter, while 2,8-di-tert-butyl-5,11-bis(4-tert-butylphenyl)-6,12-diphenyltetracene (TBRb)-doped PLQ was applied as a yellow liquid emitter. In order to form the liquid white light-emitting layer, the greenish-blue and yellow liquid emitters were alternately injected into the integrated microchannels. The fabricated electro-microfluidic device successfully exhibited white electroluminescence (EL) emission via simultaneous greenish-blue and yellow emissions under an applied voltage of 100 V. A white emission with Commission Internationale de l’Declairage (CIE) color coordinates of (0.40, 0.42) was also obtained; the emission corresponds to warm-white light. The proposed device has potential applications in subpixels of liquid-based microdisplays and for lighting.

  18. Microfluidic White Organic Light-Emitting Diode Based on Integrated Patterns of Greenish-Blue and Yellow Solvent-Free Liquid Emitters

    PubMed Central

    Kobayashi, Naofumi; Kasahara, Takashi; Edura, Tomohiko; Oshima, Juro; Ishimatsu, Ryoichi; Tsuwaki, Miho; Imato, Toshihiko; Shoji, Shuichi; Mizuno, Jun

    2015-01-01

    We demonstrated a novel microfluidic white organic light-emitting diode (microfluidic WOLED) based on integrated sub-100-μm-wide microchannels. Single-μm-thick SU-8-based microchannels, which were sandwiched between indium tin oxide (ITO) anode and cathode pairs, were fabricated by photolithography and heterogeneous bonding technologies. 1-Pyrenebutyric acid 2-ethylhexyl ester (PLQ) was used as a solvent-free greenish-blue liquid emitter, while 2,8-di-tert-butyl-5,11-bis(4-tert-butylphenyl)-6,12-diphenyltetracene (TBRb)-doped PLQ was applied as a yellow liquid emitter. In order to form the liquid white light-emitting layer, the greenish-blue and yellow liquid emitters were alternately injected into the integrated microchannels. The fabricated electro-microfluidic device successfully exhibited white electroluminescence (EL) emission via simultaneous greenish-blue and yellow emissions under an applied voltage of 100 V. A white emission with Commission Internationale de l’Declairage (CIE) color coordinates of (0.40, 0.42) was also obtained; the emission corresponds to warm-white light. The proposed device has potential applications in subpixels of liquid-based microdisplays and for lighting. PMID:26439164

  19. Astrocytes alignment and reactivity on collagen hydrogels patterned with ECM proteins

    PubMed Central

    Hsiao, Tony W.; Tresco, Patrick A.; Hlady, Vladimir

    2014-01-01

    To modulate the surface properties of collagen and subsequent cell-surface interactions, a method was developed to transfer protein patterns from glass coverslips to collagen type I hydrogel surfaces. Two proteins and one proteoglycan found in central nervous system extracellular matrix as well as fibrinogen were patterned in stripes onto collagen hydrogel and astrocytes were cultured on these surfaces. The addition of the stripe protein patterns to hydrogels created astrocyte layers in which cells were aligned with underlying patterns and had reduced chondroitin sulfate expression compared to the cells grown on collagen alone. Protein patterns were covalently cross-linked to the collagen and stable over four days in culture with no visible cellular modifications. The present method can be adapted to transfer other types of protein patterns from glass coverslips to collagen hydrogels. PMID:25477179

  20. Astrocytes alignment and reactivity on collagen hydrogels patterned with ECM proteins.

    PubMed

    Hsiao, Tony W; Tresco, Patrick A; Hlady, Vladimir

    2015-01-01

    To modulate the surface properties of collagen and subsequent cell-surface interactions, a method was developed to transfer protein patterns from glass coverslips to collagen type I hydrogel surfaces. Two proteins and one proteoglycan found in central nervous system extracellular matrix as well as fibrinogen were patterned in stripes onto collagen hydrogel and astrocytes were cultured on these surfaces. The addition of the stripe protein patterns to hydrogels created astrocyte layers in which cells were aligned with underlying patterns and had reduced chondroitin sulfate expression compared to the cells grown on collagen alone. Protein patterns were covalently cross-linked to the collagen and stable over four days in culture with no visible cellular modifications. The present method can be adapted to transfer other types of protein patterns from glass coverslips to collagen hydrogels. PMID:25477179

  1. Exploring protein-DNA interactions in 3D using in situ construction, manipulation, and visualization of individual DNA-dumbbells with optical traps, microfluidics, and fluorescence microscopy

    PubMed Central

    Forget, Anthony L.; Dombrowski, Christopher C.; Amitani, Ichiro; Kowalczykowski, Stephen C.

    2015-01-01

    In this Protocol, we describe a procedure to generate ‘DNA-dumbbells’ — single molecules of DNA with a microscopic bead attached at each end — and techniques for manipulating individual DNA-dumbbells. We also detail the design and fabrication of a microfluidic device (flow cell) used in conjunction with dual optical trapping to manipulate DNA-dumbbells and to visualize individual protein–DNA complexes by single-molecule epifluorescence microscopy. Our design of the flow cell enables the rapid movement of trapped molecules between laminar flow channels and a flow-free ‘reservoir’. The reservoir provides the means to examine formation of DNA–protein complexes in solution in the absence of external flow forces, while still maintaining a predetermined end-to-end extension of the DNA. These features facilitate examination of the role of three-dimensional DNA conformation and dynamics in protein–DNA interactions. Preparation of flow cells and reagents requires two days each; in situ DNA-dumbbell assembly and imaging of single protein–DNA complexes requires another day. PMID:23411634

  2. Trehalose glycopolymer resists allow direct writing of protein patterns by electron-beam lithography

    NASA Astrophysics Data System (ADS)

    Bat, Erhan; Lee, Juneyoung; Lau, Uland Y.; Maynard, Heather D.

    2015-03-01

    Direct-write patterning of multiple proteins on surfaces is of tremendous interest for a myriad of applications. Precise arrangement of different proteins at increasingly smaller dimensions is a fundamental challenge to apply the materials in tissue engineering, diagnostics, proteomics and biosensors. Herein, we present a new resist that protects proteins during electron-beam exposure and its application in direct-write patterning of multiple proteins. Polymers with pendant trehalose units are shown to effectively crosslink to surfaces as negative resists, while at the same time providing stabilization to proteins during the vacuum and electron-beam irradiation steps. In this manner, arbitrary patterns of several different classes of proteins such as enzymes, growth factors and immunoglobulins are realized. Utilizing the high-precision alignment capability of electron-beam lithography, surfaces with complex patterns of multiple proteins are successfully generated at the micrometre and nanometre scale without requiring cleanroom conditions.

  3. A microfluidic platform with a flow-balanced fluidic network for osteoarthritis diagnosis

    NASA Astrophysics Data System (ADS)

    Kim, Kangil; Park, Yoo Min; Yoon, Hyun C.; Yang, Sang Sik

    2013-05-01

    Osteoarthritis (OA) is one of the most common human diseases, and the occurrence of OA is likely to increase with the increase of population ages. The diagnosis of OA is based on patientrelevant measures, structural measures, and measurement of biomarkers that are released through joint metabolism. Traditionally, radiography or magnetic resonance imaging (MRI) is used to diagnose OA and predict its course. However, diagnostic imaging in OA provides only indirect information on pathology and treatment response. A sensing of OA based on the detection of biomarkers insignificantly improves the accuracy and sensitivity of diagnosis and reduces the cost compared with that of radiography or MRI. In our former study, we proposed microfluidic platform to detect biomarker of OA. But the platform can detect only one biomarker because it has one microfluidic channel. In this report, we proposes microfluidic platform that can detect several biomarkers. The proposed platform has three layers. The bottom layer has gold patterns on a Si substrate for optical sensing. The middle layer and top layer were fabricated by polydimethysiloxane (PDMS) using soft-lithography. The middle layer has four channels connecting top layer to bottom layer. The top layer consists of one sample injection inlet, and four antibody injection inlets. To this end, we designed a flow-balanced microfluidic network using analogy between electric and hydraulic systems. Also, the designed microfluidic network was confirmed by finite element model (FEM) analysis using COMSOL FEMLAB. To verify the efficiency of fabricated platform, the optical sensing test was performed to detect biomarker of OA using fluorescence microscope. We used cartilage oligomeric matrix protein (COMP) as biomarker because it reflects specific changes in joint tissues. The platform successfully detected various concentration of COMP (0, 100, 500, 1000 ng/ml) at each chamber. The effectiveness of the microfluidic platform was verified

  4. Digital Microfluidics Sample Analyzer

    NASA Technical Reports Server (NTRS)

    Pollack, Michael G.; Srinivasan, Vijay; Eckhardt, Allen; Paik, Philip Y.; Sudarsan, Arjun; Shenderov, Alex; Hua, Zhishan; Pamula, Vamsee K.

    2010-01-01

    Three innovations address the needs of the medical world with regard to microfluidic manipulation and testing of physiological samples in ways that can benefit point-of-care needs for patients such as premature infants, for which drawing of blood for continuous tests can be life-threatening in their own right, and for expedited results. A chip with sample injection elements, reservoirs (and waste), droplet formation structures, fluidic pathways, mixing areas, and optical detection sites, was fabricated to test the various components of the microfluidic platform, both individually and in integrated fashion. The droplet control system permits a user to control droplet microactuator system functions, such as droplet operations and detector operations. Also, the programming system allows a user to develop software routines for controlling droplet microactuator system functions, such as droplet operations and detector operations. A chip is incorporated into the system with a controller, a detector, input and output devices, and software. A novel filler fluid formulation is used for the transport of droplets with high protein concentrations. Novel assemblies for detection of photons from an on-chip droplet are present, as well as novel systems for conducting various assays, such as immunoassays and PCR (polymerase chain reaction). The lab-on-a-chip (a.k.a., lab-on-a-printed-circuit board) processes physiological samples and comprises a system for automated, multi-analyte measurements using sub-microliter samples of human serum. The invention also relates to a diagnostic chip and system including the chip that performs many of the routine operations of a central labbased chemistry analyzer, integrating, for example, colorimetric assays (e.g., for proteins), chemiluminescence/fluorescence assays (e.g., for enzymes, electrolytes, and gases), and/or conductometric assays (e.g., for hematocrit on plasma and whole blood) on a single chip platform.

  5. Instability mechanisms in microfluidics and nanomaterials

    NASA Astrophysics Data System (ADS)

    Thamida, Sunil Kumar

    -organization dynamics of the pores. In microfluidic devices, electrokinetic flow produces spiral vortices and corner aggregation of particles and proteins at an inner corner of a channel turn that is unexplained by the short ranged DLVO forces. Field leakage effect due to the non perfectly insulating wall reveals a nonlinear singular and ejecting slip velocity condition at an acute angled sharp corner. The complete flow streamlines, vortices and the corner entrainment are revealed by conformal mapping, harmonic analysis and numerical simulation using Lattice-Boltzmann-Method (LBM). The method of hodograph transform developed for the earlier projects to solve the Laplace equation is also applied to find optimum shapes of dispersion free turns for electro-osmotic microfluidic channels.

  6. Easy Fabrication of Thin Membranes with Through Holes. Application to Protein Patterning

    PubMed Central

    Arasi, Bakya; Gauthier, Nils; Viasnoff, Virgile

    2012-01-01

    Since protein patterning on 2D surfaces has emerged as an important tool in cell biology, the development of easy patterning methods has gained importance in biology labs. In this paper we present a simple, rapid and reliable technique to fabricate thin layers of UV curable polymer with through holes. These membranes are as easy to fabricate as microcontact printing stamps and can be readily used for stencil patterning. We show how this microfabrication scheme allows highly reproducible and highly homogeneous protein patterning with micron sized resolution on surfaces as large as 10 cm2. Using these stencils, fragile proteins were patterned without loss of function in a fully hydrated state. We further demonstrate how intricate patterns of multiple proteins can be achieved by stacking the stencil membranes. We termed this approach microserigraphy. PMID:22952944

  7. Easy fabrication of thin membranes with through holes. Application to protein patterning.

    PubMed

    Masters, Thomas; Engl, Wilfried; Weng, Zhe L; Arasi, Bakya; Gauthier, Nils; Viasnoff, Virgile

    2012-01-01

    Since protein patterning on 2D surfaces has emerged as an important tool in cell biology, the development of easy patterning methods has gained importance in biology labs. In this paper we present a simple, rapid and reliable technique to fabricate thin layers of UV curable polymer with through holes. These membranes are as easy to fabricate as microcontact printing stamps and can be readily used for stencil patterning. We show how this microfabrication scheme allows highly reproducible and highly homogeneous protein patterning with micron sized resolution on surfaces as large as 10 cm(2). Using these stencils, fragile proteins were patterned without loss of function in a fully hydrated state. We further demonstrate how intricate patterns of multiple proteins can be achieved by stacking the stencil membranes. We termed this approach microserigraphy. PMID:22952944

  8. Patterned polymer nanowire arrays as an effective protein immobilizer for biosensing and HIV detection

    NASA Astrophysics Data System (ADS)

    Shen, Yue; Liu, Yingyi; Zhu, Guang; Fang, Hao; Huang, Yunhui; Jiang, Xingyu; Wang, Zhong L.

    2012-12-01

    We report an array of polymeric nanowires for effectively immobilizing biomolecules on biochips owing to the large surface area. The nanowires were fabricated in predesigned patterns using an inductively coupled plasma (ICP) etching process. Microfluidic biochips integrated using the substrates with arrays of nanowires and polydimethylsiloxane channels have been demonstrated to be effective for detecting antigens, and a detection limit of antigens at 0.2 μg mL-1 has been achieved, which is improved by a factor of 50 compared to that based on flat substrates without the nanowires. In addition, the high sensitivity for clinical detection of human immunodeficiency virus (HIV) antibody has also been demonstrated, showing a 20 times enhancement in fluorescent signal intensity between the samples with positive and negative HIV.

  9. Patterned polymer nanowire arrays as an effective protein immobilizer for biosensing and HIV detection.

    PubMed

    Shen, Yue; Liu, Yingyi; Zhu, Guang; Fang, Hao; Huang, Yunhui; Jiang, Xingyu; Wang, Zhong L

    2013-01-21

    We report an array of polymeric nanowires for effectively immobilizing biomolecules on biochips owing to the large surface area. The nanowires were fabricated in predesigned patterns using an inductively coupled plasma (ICP) etching process. Microfluidic biochips integrated using the substrates with arrays of nanowires and polydimethylsiloxane channels have been demonstrated to be effective for detecting antigens, and a detection limit of antigens at 0.2 μg mL(-1) has been achieved, which is improved by a factor of 50 compared to that based on flat substrates without the nanowires. In addition, the high sensitivity for clinical detection of human immunodeficiency virus (HIV) antibody has also been demonstrated, showing a 20 times enhancement in fluorescent signal intensity between the samples with positive and negative HIV. PMID:23223639

  10. Predicting Droplet Formation on Centrifugal Microfluidic Platforms

    NASA Astrophysics Data System (ADS)

    Moebius, Jacob Alfred

    Centrifugal microfluidics is a widely known research tool for biological sample and water quality analysis. Currently, the standard equipment used for such diagnostic applications include slow, bulky machines controlled by multiple operators. These machines can be condensed into a smaller, faster benchtop sample-to-answer system. Sample processing is an important step taken to extract, isolate, and convert biological factors, such as nucleic acids or proteins, from a raw sample to an analyzable solution. Volume definition is one such step. The focus of this thesis is the development of a model predicting monodispersed droplet formation and the application of droplets as a technique for volume definition. First, a background of droplet microfluidic platforms is presented, along with current biological analysis technologies and the advantages of integrating such technologies onto microfluidic platforms. Second, background and theories of centrifugal microfluidics is given, followed by theories relevant to droplet emulsions. Third, fabrication techniques for centrifugal microfluidic designs are discussed. Finally, the development of a model for predicting droplet formation on the centrifugal microfluidic platform are presented for the rest of the thesis. Predicting droplet formation analytically based on the volumetric flow rates of the continuous and dispersed phases, the ratios of these two flow rates, and the interfacial tension between the continuous and dispersed phases presented many challenges, which will be discussed in this work. Experimental validation was completed using continuous phase solutions of different interfacial tensions. To conclude, prospective applications are discussed with expected challenges.