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Sample records for lipid-based protein immobilization

  1. Elucidation of the outer membrane proteome of Salmonella enterica serovar Typhimurium utilising a lipid-based protein immobilization technique

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    Appleton Hazel

    2010-02-01

    Full Text Available Abstract Background Salmonella enterica serovar Typhimurium (S. Typhimurium is a major cause of human gastroenteritis worldwide. The outer membrane proteins expressed by S. Typhimurium mediate the process of adhesion and internalisation within the intestinal epithelium of the host thus influencing the progression of disease. Since the outer membrane proteins are surface-exposed, they provide attractive targets for the development of improved antimicrobial agents and vaccines. Various techniques have been developed for their characterisation, but issues such as carryover of cytosolic proteins still remain a problem. In this study we attempted to characterise the surface proteome of S. Typhimurium using Lipid-based Protein Immobilisation technology in the form of LPI™ FlowCells. No detergents are required and no sample clean up is needed prior to downstream analysis. The immobilised proteins can be digested with proteases in multiple steps to increase sequence coverage, and the peptides eluted can be characterised directly by liquid chromatography - tandem mass spectrometry (LC-MS/MS and identified from mass spectral database searches. Results In this study, 54 outer membrane proteins, were identified with two or more peptide hits using a multi-step digest approach. Out of these 28 were lipoproteins, nine were involved in transport and three with enzyme activity These included the transporters BtuB which is responsible for the uptake of vitamin B12, LamB which is involved in the uptake of maltose and maltodextrins and LolB which is involved in the incorporation of lipoproteins in the outer membrane. Other proteins identified included the enzymes MltC which may play a role in cell elongation and division and NlpD which is involved in catabolic processes in cell wall formation as well as proteins involved in virulence such as Lpp1, Lpp2 and OmpX. Conclusion Using a multi-step digest approach the LPI™ technique enables the incorporation of a

  2. Oral delivery of peptides and proteins using lipid-based drug delivery systems

    DEFF Research Database (Denmark)

    Li, Ping; Nielsen, Hanne Mørck; Müllertz, Anette

    2012-01-01

    INTRODUCTION: In order to successfully develop lipid-based drug delivery systems (DDS) for oral administration of peptides and proteins, it is important to gain an understanding of the colloid structures formed by these DDS, the mode of peptide and protein incorporation as well as the mechanism...... by which intestinal absorption of peptides and proteins is promoted. AREAS COVERED: The present paper reviews the literature on lipid-based DDS, employed for oral delivery of peptides and proteins and highlights the mechanisms by which the different lipid-based carriers are expected to overcome the two...... and proteins. EXPERT OPINION: Lipid-based DDS are safe and suitable for oral delivery of peptides and proteins. Significant progress has been made in this area with several technologies on clinical trials. However, a better understanding of the mechanism of action in vivo is needed in order to improve...

  3. Supramolecular protein immobilization on lipid bilayers

    NARCIS (Netherlands)

    Bosmans, R.P.G.; Hendriksen, W.E.; Verheijden, Mark Lloyd; Eelkema, R.; Jonkheijm, Pascal; van Esch, J.H.; Brunsveld, Luc

    2015-01-01

    Protein immobilization on surfaces, and on lipid bilayers specifically, has great potential in biomolecular and biotechnological research. Of current special interest is the immobilization of proteins using supramolecular noncovalent interactions. This allows for a reversible immobilization and

  4. Oral delivery of peptides and proteins using lipid-based drug delivery systems.

    Science.gov (United States)

    Li, Ping; Nielsen, Hanne Mørck; Müllertz, Anette

    2012-10-01

    In order to successfully develop lipid-based drug delivery systems (DDS) for oral administration of peptides and proteins, it is important to gain an understanding of the colloid structures formed by these DDS, the mode of peptide and protein incorporation as well as the mechanism by which intestinal absorption of peptides and proteins is promoted. The present paper reviews the literature on lipid-based DDS, employed for oral delivery of peptides and proteins and highlights the mechanisms by which the different lipid-based carriers are expected to overcome the two most important barriers (extensive enzymatic degradation and poor transmucosal permeability). This paper also gives a clear-cut idea about advantages and drawbacks of using different lipidic colloidal carriers ((micro)emulsions, solid lipid core particles and liposomes) for oral delivery of peptides and proteins. Lipid-based DDS are safe and suitable for oral delivery of peptides and proteins. Significant progress has been made in this area with several technologies on clinical trials. However, a better understanding of the mechanism of action in vivo is needed in order to improve the design and development of lipid-based DDS with the desired bioavailability and therapeutic profile.

  5. Strong and Reversible Monovalent Supramolecular Protein Immobilization

    NARCIS (Netherlands)

    Young, Jacqui F.; Nguyen, Hoang D.; Yang, Lanti; Huskens, Jurriaan; Jonkheijm, Pascal; Brunsveld, Luc

    2010-01-01

    Proteins with an iron clasp: Site-selective incorporation of a ferrocene molecule into a protein allows for easy, strong, and reversible supramolecular protein immobilization through a selective monovalent interaction of the ferrocene with a cucurbit[7]uril immobilized on a gold surface. The

  6. Protein immobilization strategies for protein biochips

    NARCIS (Netherlands)

    Rusmini, F.; Rusmini, Federica; Zhong, Zhiyuan; Feijen, Jan

    2007-01-01

    In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein

  7. Protein synthesis rates in atrophied gastrocnemius muscles after limb immobilization

    Science.gov (United States)

    Tucker, K. R.; Seider, M. J.; Booth, F. W.

    1981-01-01

    Noting that protein synthesis declines in the gastrocnemius 6 hr after immobilization, the study sought to detect an increase of protein synthesis when the limb was freed, and to examine the effects of exercise on the rate of increase. Rats were used as subjects, with their hind legs in plaster of Paris in plantar flexion to eliminate strain on the gastrocnemius. Periods of immobilization were varied and samples of blood from the muscle were taken to track protein synthesis rates for different groups in immobilization and exercise regimens (running and weightlifting). Synthesis rates declined 3.6% during time in the cast, then increased 6.3%/day after the casts were removed. Both running and weightlifting were found to increase the fractional rate of protein formation in the gastrocnemius muscle when compared with contralateral muscles that were not exercised and were used as controls, suggesting that the mechanism controlling protein synthesis in skeletal muscles is rapidly responsive to changes in muscular contractile activity.

  8. {sup 125}I Labelling of Protein Using Immobilized Enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jae Rok; Park, Kyung Bae; Awh, Ok Doo [Korea Advanced Energy Research Institute, Daejeon (Korea, Republic of)

    1984-03-15

    For an effective solid-phase labelling of protein with {sup 125}I, studies on the immobilization of lactoperoxidase (LPO) on the inner wall of polystyrene tubes were carried out. Labelling of bovine serum albumin (BSA) and insulin was also practiced using the LPO immobilized tubes. The immobilized enzyme of about 2.5 mu g/tube was sufficient for small scale labelling since the results of radio-paper chromatography of the labelling mixture of insulin indicated that the yields were sufficiently high (80%) even in the reactions conducted at room temperature for 60 sec. The results of the Sephadex column chromatography indicated that the labelled products were not contaminated with LPO-{sup 125}I, and the radiochemical purity of the products was more than 90%. In considering the general trend that the {sup 125}I labelled protein obtained by using LPO maintains its intactness better than those obtained by using chloramine-T, together with the tendency of yield enhancing with increase of reactants-concentration, the LPO immobilized tube method is estimated to be one of the simple methods of labelling. The product might be applicable without further purification.

  9. Determination of conformation and orientation of immobilized peptides and proteins at buried interfaces

    Science.gov (United States)

    Shen, Lei; Ulrich, Nathan W.; Mello, Charlene M.; Chen, Zhan

    2015-01-01

    Surface immobilized peptides/proteins have important applications such as antimicrobial coating and biosensing. We report a study of such peptides/proteins using sum frequency generation vibrational spectroscopy and ATR-FTIR. Immobilization on surfaces via physical adsorption and chemical coupling revealed that structures of chemically immobilized peptides are determined by immobilization sites, chemical environments, and substrate surfaces. In addition, controlling enzyme orientation by engineering the surface immobilization site demonstrated that structures can be well-correlated to measured chemical activity. This research facilitates the development of immobilized peptides/proteins with improved activities by optimizing their surface orientation and structure.

  10. Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

    Science.gov (United States)

    Cha, Jaehyun; Kwon, Inchan

    2018-02-27

    Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Binary polypeptide system for permanent and oriented protein immobilization

    Directory of Open Access Journals (Sweden)

    Bailes Julian

    2010-05-01

    Full Text Available Abstract Background Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag. Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case leading to the requirement for chemical coupling. Results Here we report a new immobilization system which utilizes two monomeric polypeptides which self-assemble to produce non-covalent yet nearly irreversible complex which is stable in strong detergents, chaotropic agents, as well as in acids and alkali. Our system is based on the core region of the tetra-helical bundle known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex. This irreversible protein attachment system (IPAS uses either a shortened syntaxin helix and fused SNAP25-synaptobrevin or a fused syntaxin-synaptobrevin and SNAP25 allowing a two-component system suitable for recombinant protein tagging, capture and immobilization. We also show that IPAS is suitable for use with traditional beads and chromatography, planar surfaces and Biacore, gold nanoparticles and for protein-protein interaction in solution. Conclusions IPAS offers an alternative to chemical cross-linking, streptavidin-biotin system and to traditional peptide affinity tags and can be used for a wide range of applications in nanotechnology and molecular sciences.

  12. Novel humic acid-bonded magnetite nanoparticles for protein immobilization

    Energy Technology Data Exchange (ETDEWEB)

    Bayrakci, Mevlut, E-mail: mevlutbayrakci@gmail.com [Ulukisla Vocational School, Nigde University, 51100 Ulukisla, Nigde (Turkey); Gezici, Orhan [Department of Chemistry, Nigde University, 51100 Nigde (Turkey); Bas, Salih Zeki; Ozmen, Mustafa; Maltas, Esra [Department of Chemistry, Selcuk University, 42031 Konya (Turkey)

    2014-09-01

    The present paper is the first report that introduces (i) a useful methodology for chemical immobilization of humic acid (HA) to aminopropyltriethoxysilane-functionalized magnetite iron oxide nanoparticles (APS-MNPs) and (ii) human serum albumin (HSA) binding to the obtained material (HA-APS-MNPs). The newly prepared magnetite nanoparticle was characterized by using Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and elemental analysis. Results indicated that surface modification of the bare magnetite nanoparticles (MNPs) with aminopropyltriethoxysilane (APS) and HA was successfully performed. The protein binding studies that were evaluated in batch mode exhibited that HA-APS-MNPs could be efficiently used as a substrate for the binding of HSA from aqueous solutions. Usually, recovery values higher than 90% were found to be feasible by HA-APS-MNPs, while that value was around 2% and 70% in the cases of MNPs and APS-MNPs, respectively. Hence, the capacity of MNPs was found to be significantly improved by immobilization of HA. Furthermore, thermal degradation of HA-APS-MNPs and HSA bonded HA-APS-MNPs was evaluated in terms of the Horowitz–Metzger equation in order to determine kinetic parameters for thermal decomposition. Activation energies calculated for HA-APS-MNPs (20.74 kJ mol{sup −1}) and HSA bonded HA-APS-MNPs (33.42 kJ mol{sup −1}) implied chemical immobilization of HA to APS-MNPs, and tight interactions between HA and HA-APS-MNPs. - Highlights: • A new magnetite nanoparticle based humic acid was prepared for the first time. • Protein binding studies of magnetite nanoparticle based humic acid were performed. • Kinetic parameters of protein and/or humic acid bonded nanoparticles were evaluated.

  13. Recent advances in covalent, site-specific protein immobilization [version 1; referees

    DEFF Research Database (Denmark)

    Meldal, Morten Peter; Schoffelen, Sanne

    2016-01-01

    The properties of biosensors, biomedical implants, and other materials based on immobilized proteins greatly depend on the method employed to couple the protein molecules to their solid support. Covalent, site-specific immobilization strategies are robust and can provide the level of control...

  14. Protein selectivity with immobilized metal ion-tacn sorbents: chromatographic studies with human serum proteins and several other globular proteins.

    Science.gov (United States)

    Jiang, W; Graham, B; Spiccia, L; Hearn, M T

    1998-01-01

    The chromatographic selectivity of the immobilized chelate system, 1,4,7-triazocyclononane (tacn), complexed with the borderline metal ions Cu2+, Cr3+, Mn2+, Co2+, Zn2+, and Ni2+ has been investigated with hen egg white lysozyme, horse heart cytochrome c, and horse skeletal muscle myoglobin, as well as proteins present in partially fractionated preparations of human plasma. The effects of ionic strength and pH of the loading and elution buffers on protein selectivities of these new immobilized metal ion affinity chromatographic (IMAC) systems have been examined. The results confirm that immobilized Mn;pl-tacn sorbents exhibit a novel type of IMAC behavior with proteins. In particular, the chromatographic properties of these immobilized M(n+)-tacn ligand systems were significantly different compared to the IMAC behavior observed with other types of immobilized tri- and tetradentate chelating ligands, such as iminodiacetic acid, O-phosphoserine, or nitrilotriacetic acid, when complexed with borderline metal ions. The experimental results have consequently been evaluated in terms of the additional contributions to the interactive processes mediated by effects other than solely the conventional lone pair Lewis soft acid-Lewis soft base coordination interactions, typically found for the IMAC of proteins with borderline and soft metal ions, such as Cu2+ or Ni2+.

  15. Hydrolysis of fish protein by Bacillus megaterium cells immobilized in radiation induced polymerized wood

    International Nuclear Information System (INIS)

    Ghosh, S.; Alur, M.D.; Nerkar, D.P.

    1992-01-01

    The immobilization of Bacillus megaterium cells in radiation-induced polymerized wood was studied for hydrolysis of trash fish protein. The optimum conditions and reaction kinetics for hydrolysis of protein by free and immobilized cells were found to be similar. Maximum hydrolysis occurred at 50 o C and at pH 7.5 with 15-20% (w/v) of immobilized matrix. The soluble content of the resultant hydrolysate about 2.4% (w/v). (author). 10 refs., 4 figs

  16. Improvement of the homogeneity of protein-imprinted polymer films by orientated immobilization of the template

    International Nuclear Information System (INIS)

    Liu Lijian; Zheng Jingjing; Fang Guijie; Xie Weihong

    2012-01-01

    Highlights: ► MPH was genetically modified at its C-terminal with (Gly-Ser) 5 –Cys. ► MPH-L was immobilized with fixed orientation via disulfide chemistry. ► The immobilized MPH-L retained the activity of MPH. ► MPH-L formed a homogeneous template. ► Homogeneous MIP film was obtained with orientated immobilization of the template. - Abstract: A method for preparing homogeneous protein-imprinted polymer films with orientated immobilization of template is described. The template methyl parathion hydrolase (MPH) was modified with a peptide linker (Gly-Ser) 5 –Cys and was immobilized on a cover glass with a fixed orientation via the linker. The activity of the fusion enzyme (MPH-L) was evaluated by determining the product's absorbance at 405 nm (A 405 ). Both the free and the immobilized MPH-L showed higher retention of the bioactivity than the wide type enzyme (MPH-W) as revealed by the A 405 values for MPH-L free /MPH-W free (1.159/1.111) and for MPH-L immobilized /MPH-W immobilized (0.348/0.118). The immobilized MPH-L also formed a more homogeneous template stamp compared to the immobilized MPH-W. The molecularly imprinted polymer films prepared with the immobilized MPH-L exhibited high homogeneity with low Std. Deviations of 80 and 200 from the CL intensity mean volumes which were observed for batch-prepared films and an individual film, respectively. MPH-L-imprinted polymer film also had a larger template binding capacity indicated by higher CL intensity mean volume of 3900 INT over 2500 INT for MPH-W-imprinted films. The imprinted film prepared with the orientated immobilization of template showed an imprinting factor of 1.7, while the controls did not show an imprinting effect.

  17. Site-directed antibody immobilization using a protein A-gold binding domain fusion protein for enhanced SPR immunosensing.

    Science.gov (United States)

    de Juan-Franco, Elena; Caruz, Antonio; Pedrajas, J R; Lechuga, Laura M

    2013-04-07

    We have implemented a novel strategy for the oriented immobilization of antibodies onto a gold surface based on the use of a fusion protein, the protein A-gold binding domain (PAG). PAG consists of a gold binding peptide (GBP) coupled to the immunoglobulin-binding domains of staphylococcal protein A. This fusion protein provides an easy and fast oriented immobilization of antibodies preserving its native structure, while leaving the antigen binding sites (Fab) freely exposed. Using this immobilization strategy, we have demonstrated the performance of the immunosensing of the human Growth Hormone by SPR. A limit of detection of 90 ng mL(-1) was obtained with an inter-chip variability lower than 7%. The comparison of this method with other strategies for the direct immobilization of antibodies over gold surfaces has showed the enhanced sensitivity provided by the PAG approach.

  18. Biospecific protein immobilization for rapid analysis of weak protein interactions using self-interaction nanoparticle spectroscopy.

    Science.gov (United States)

    Bengali, Aditya N; Tessier, Peter M

    2009-10-01

    "Reversible" protein interactions govern diverse biological behavior ranging from intracellular transport and toxic protein aggregation to protein crystallization and inactivation of protein therapeutics. Much less is known about weak protein interactions than their stronger counterparts since they are difficult to characterize, especially in a parallel format (in contrast to a sequential format) necessary for high-throughput screening. We have recently introduced a highly efficient approach of characterizing protein self-association, namely self-interaction nanoparticle spectroscopy (SINS; Tessier et al., 2008; J Am Chem Soc 130:3106-3112). This approach exploits the separation-dependent optical properties of gold nanoparticles to detect weak self-interactions between proteins immobilized on nanoparticles. A limitation of our previous work is that differences in the sequence and structure of proteins can lead to significant differences in their affinity to adsorb to nanoparticle surfaces, which complicates analysis of the corresponding protein self-association behavior. In this work we demonstrate a highly specific approach for coating nanoparticles with proteins using biotin-avidin interactions to generate protein-nanoparticle conjugates that report protein self-interactions through changes in their optical properties. Using lysozyme as a model protein that is refractory to characterization by conventional SINS, we demonstrate that surface Plasmon wavelengths for gold-avidin-lysozyme conjugates over a range of solution conditions (i.e., pH and ionic strength) are well correlated with lysozyme osmotic second virial coefficient measurements. Since SINS requires orders of magnitude less protein and time than conventional methods (e.g., static light scattering), we envision this approach will find application in large screens of protein self-association aimed at either preventing (e.g., protein aggregation) or promoting (e.g., protein crystallization) these

  19. Recent Developments in the Site-Specific Immobilization of Proteins onto Solid Supports

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A

    2007-02-21

    Immobilization of proteins onto surfaces is of great importance in numerous applications, including protein analysis, drug screening, and medical diagnostics, among others. The success of all these technologies relies on the immobilization technique employed to attach a protein to the corresponding surface. Non-specific physical adsorption or chemical cross-linking with appropriate surfaces results in the immobilization of the protein in random orientations. Site-specific covalent attachment, on the other hand, leads to molecules being arranged in a definite, orderly fashion and allows the use of spacers and linkers to help minimize steric hindrances between the protein and the surface. The present work reviews the latest chemical and biochemical developments for the site-specific covalent attachment of proteins onto solid supports.

  20. Orented immobilization of farnesylated proteins by the thiol-ene reaction

    NARCIS (Netherlands)

    Weinrich, Dirk; Lin, Po-Chiao; Jonkheijm, Pascal; Nguyen, Uyen T.T.; Schröder, Hendrik; Niemeyer, Christof M.; Alexandrov, Kirill; Goody, Roger; Waldmann, Herbert

    2010-01-01

    Anchoring the protein: Proteins were immobilized rapidly under mild conditions by thiol-ene photocoupling between S-farnesyl groups attached to a genetically encodable “CAAX-box” tetrapeptide sequence (A is aliphatic) at the C terminus of the protein and surface-exposed thiols (see scheme). This

  1. An orientation analysis method for protein immobilized on quantum dot particles

    Energy Technology Data Exchange (ETDEWEB)

    Aoyagi, Satoka, E-mail: aoyagi@life.shimane-u.ac.jp [Faculty of Life and Environmental Science, Shimane University, 1060 Matsue-shi, Shimane 690-8504 (Japan); Inoue, Masae [Toyota Central R and D Labs., Inc., Nagakute, Aichi 480-1192 (Japan)

    2009-11-30

    The evaluation of orientation of biomolecules immobilized on nanodevices is crucial for the development of high performance devices. Such analysis requires ultra high sensitivity so as to be able to detect less than one molecular layer on a device. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has sufficient sensitivity to evaluate the uppermost surface structure of a single molecular layer. The objective of this study is to develop an orientation analysis method for proteins immobilized on nanomaterials such as quantum dot particles, and to evaluate the orientation of streptavidin immobilized on quantum dot particles by means of TOF-SIMS. In order to detect fragment ions specific to the protein surface, a monoatomic primary ion source (Ga{sup +}) and a cluster ion source (Au{sub 3}{sup +}) were employed. Streptavidin-immobilized quantum dot particles were immobilized on aminosilanized ITO glass plates at amino groups by covalent bonding. The reference samples streptavidin directly immobilized on ITO plates were also prepared. All samples were dried with a freeze dryer before TOF-SIMS measurement. The positive secondary ion spectra of each sample were obtained using TOF-SIMS with Ga{sup +} and Au{sub 3}{sup +}, respectively, and then they were compared so as to characterize each sample and detect the surface structure of the streptavidin immobilized with the biotin-immobilized quantum dots. The chemical structures of the upper surface of the streptavidin molecules immobilized on the quantum dot particles were evaluated with TOF-SIMS spectra analysis. The indicated surface side of the streptavidin molecules immobilized on the quantum dots includes the biotin binding site.

  2. Reaching (sub-)micrometer resolution of photo-immobilized proteins using diffracted light beams

    DEFF Research Database (Denmark)

    Skovsen, Esben; Neves Petersen, Teresa; Petersen, Steffen B.

    2008-01-01

    , with dimensions as small as a few micrometers. The ultimate size of the immobilized spots is dependent on the focal area of the UV beam. The technology involves light induced formation of free, reactive thiol groups in molecules containing aromatic residues nearby disulphide bridges. It is not only limited...... to immobilizing molecules according to conventional patterns like microarrays, as any bitmap motif can virtually be used a template for patterning. We now show that molecules (proteins) can be immobilized on a surface with any arbitrary pattern according to diffraction patterns of light. The pattern of photo......-immobilized proteins reproduces the diffraction pattern of light expected with the optical setup. Immobilising biomolecules according to diffraction patterns of light will allow achievement of smaller patterns with higher resolution. The flexibility of this new technology leads to any patterns of photo...

  3. Facile and high-efficient immobilization of histidine-tagged multimeric protein G on magnetic nanoparticles

    Science.gov (United States)

    Lee, Jiho; Chang, Jeong Ho

    2014-12-01

    This work reports the high-efficient and one-step immobilization of multimeric protein G on magnetic nanoparticles. The histidine-tagged (His-tag) recombinant multimeric protein G was overexpressed in Escherichia coli BL21 by the repeated linking of protein G monomers with a flexible linker. High-efficient immobilization on magnetic nanoparticles was demonstrated by two different preparation methods through the amino-silane and chloro-silane functionalization on silica-coated magnetic nanoparticles. Three kinds of multimeric protein G such as His-tag monomer, dimer, and trimer were tested for immobilization efficiency. For these tests, bicinchoninic acid (BCA) assay was employed to determine the amount of immobilized His-tag multimeric protein G. The result showed that the immobilization efficiency of the His-tag multimeric protein G of the monomer, dimer, and trimer was increased with the use of chloro-silane-functionalized magnetic nanoparticles in the range of 98% to 99%, rather than the use of amino-silane-functionalized magnetic nanoparticles in the range of 55% to 77%, respectively.

  4. Quantitative electrical detection of immobilized protein using gold nanoparticles and gold enhancement on a biochip

    International Nuclear Information System (INIS)

    Lei, Kin Fong

    2011-01-01

    Electrical detection of the concentration of protein immobilized on a biochip is demonstrated. The concentration of the direct immobilized protein can be determined by the resistance values measured by an ohm-meter directly. Indium tin oxide interdigitated electrodes were utilized as the detection sites on the biochip. Protein, i.e. antibody, of certain concentration was first immobilized on the detection site. Gold nanoparticles were then applied to indicate the immobilized protein. Since the gold nanoparticles were tiny, a detectable electrical signal could not be generated. Hence, a gold enhancement process was performed for signal amplification. Gold nanoparticles were enlarged physically, such that a conductive metal layer was formed on the detection site. The presence and concentration of protein can be determined by the resistance value across the electrode measured by an ohm-meter. An immobilized protein concentration ranging from 50 to 1000 ng ml −1 can be detected quantitatively by the resistance values from 4300 to 1700 Ω. The proposed technique is potentially extended for the detection of immunoassay on the biochip. Since the protocol of the electrical detection does not involve sophisticated equipment, it can therefore be used for the development of a portable immunoassay device

  5. Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers.

    Science.gov (United States)

    Holstein, Carly A; Chevalier, Aaron; Bennett, Steven; Anderson, Caitlin E; Keniston, Karen; Olsen, Cathryn; Li, Bing; Bales, Brian; Moore, David R; Fu, Elain; Baker, David; Yager, Paul

    2016-02-01

    To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.

  6. Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients

    NARCIS (Netherlands)

    Bischoff, Rainer; Roecklin, D.; Roitsch, C.

    1992-01-01

    Isoelectric focusing in immobilized pH gradients (IEF-IPG) was used to analyze three different recombinant proteins. Recombinant leech hirudin (65 amino acids, three disulfide bonds) expressed in Saccharomyces cerevisiae as a secreted protein and purified by anion-exchange and reversed-phase

  7. Evaluation of protein immobilization capacity on various carbon nanotube embedded hydrogel biomaterials

    Energy Technology Data Exchange (ETDEWEB)

    Derkus, Burak, E-mail: burakderkus@gmail.com; Emregul, Kaan Cebesoy; Emregul, Emel

    2015-11-01

    This study investigates effective immobilization of proteins, an important procedure in many fields of bioengineering and medicine, using various biomaterials. Gelatin, alginate and chitosan were chosen as polymeric carriers, and applied in both their composites and nanocomposite forms in combination with carbon nanotubes (CNTs). The prepared nano/composite structures were characterized using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TG) and contact angle analysis (CA). Electrochemical impedance spectroscopy analysis revealed gelatin composites in general to exhibit better immobilization performance relative to the native gelatin which can be attributed to enhanced film morphologies of the composite structures. Moreover, superior immobilization efficiencies were obtained with the addition of carbon nanotubes, due to their conducting and surface enhancement features, especially in the gelatin–chitosan structures due to the presence of structural active groups. - Highlights: • Various nanocomposite biomaterials were developed for efficient immobilization of proteins. • CNTs enhance the immobilization efficiency owing to their conducting and surface enhancement features. • Gelatin–chitosan–CNTs structure is promising immobilization matrix thanks to its effective CNTs binding capacity.

  8. Analysis of direct immobilized recombinant protein G on a gold surface

    International Nuclear Information System (INIS)

    Kim, Hyunhee; Kang, Da-Yeon; Goh, Hyun-Jeong; Oh, Byung-Keun; Singh, Ravindra P.; Oh, Soo-Min; Choi, Jeong-Woo

    2008-01-01

    Abstact: For the immobilization of IgG, various techniques such as chemical linker, thiolated protein G methods, and fragmentation of antibodies have been reported [Y.M. Bae, B.K. Oh, W. Lee, W.H. Lee, J.W. Choi, Biosensors Bioelectron. 21 (2005) 103; W. Lee, B.K. Oh, W.H. Lee, J.W. Choi, Colloids Surf. B-Biointerfaces, 40 (2005) 143; A.A. Karyakin, G.V. Presnova, M.Y. Rubtsova, A.M. Egorov, Anal. Chem. 72 (2000) 3805]. Here, we modified the immunoglobulin Fc-binding B-domain of protein G to contain two cysteine residues at its C-terminus by a genetic engineering technique. The resulting recombinant protein, RPGcys, retained IgG-binding activity in the same manner as native protein G. RPGcys was immobilized on a gold surface by strong affinity between thiol of cysteine and gold. The orientations of both IgG layers immobilized on the base recombinant protein Gs were analyzed by fluorescence microscope, atomic force microscope (AFM), and surface plasmon resonance (SPR). Our data revealed that IgG-binding activity of RPGcys on gold surface significantly increased in comparison to wild type of protein G (RPGwild), which was physically adsorbed due to absence of cysteine residue. Immobilization of highly oriented antibodies based on cysteine-modified protein G could be useful for the fabrication of immunosensor systems

  9. Immobilization-stabilization of proteins on nanofibrillated cellulose derivatives and their bioactive film formation.

    Science.gov (United States)

    Arola, Suvi; Tammelin, Tekla; Setälä, Harri; Tullila, Antti; Linder, Markus B

    2012-03-12

    In a number of different applications for enzymes and specific binding proteins a key technology is the immobilization of these proteins to different types of supports. In this work we describe a concept for protein immobilization that is based on nanofibrillated cellulose (NFC). NFC is a form of cellulose where fibers have been disintegrated into fibrils that are only a few nanometers in diameter and have a very large aspect ratio. Proteins were conjugated through three different strategies using amine, epoxy, and carboxylic acid functionalized NFC. The conjugation chemistries were chosen according to the reactive groups on the NFC derivatives; epoxy amination, heterobifunctional modification of amino groups, and EDC/s-NHS activation of carboxylic acid groups. The conjugation reactions were performed in solution and immobilization was performed by spin coating the protein-NCF conjugates. The structure of NFC was shown to be advantageous for both protein performance and stability. The use of NFC allows all covalent chemistry to be performed in solution, while the immobilization is achieved by a simple spin coating or spreading of the protein-NFC conjugates on a support. This allows more scalable methods and better control of conditions compared to the traditional methods that depend on surface reactions.

  10. Co-Immobilization of Proteins and DNA Origami Nanoplates to Produce High-Contrast Biomolecular Nanoarrays.

    Science.gov (United States)

    Hager, Roland; Burns, Jonathan R; Grydlik, Martyna J; Halilovic, Alma; Haselgrübler, Thomas; Schäffler, Friedrich; Howorka, Stefan

    2016-06-01

    The biofunctionalization of nanopatterned surfaces with DNA origami nanostructures is an important topic in nanobiotechnology. An unexplored challenge is, however, to co-immobilize proteins with DNA origami at pre-determined substrate sites in high contrast relative to the nontarget areas. The immobilization should, in addition, preferably be achieved on a transparent substrate to allow ultrasensitive optical detection. If successful, specific co-binding would be a step towards stoichiometrically defined arrays with few to individual protein molecules per site. Here, we successfully immobilize with high specificity positively charged avidin proteins and negatively charged DNA origami nanoplates on 100 nm-wide carbon nanoislands while suppressing undesired adsorption to surrounding nontarget areas. The arrays on glass slides achieve unprecedented selectivity factors of up to 4000 and allow ultrasensitive fluorescence read-out. The co-immobilization onto the nanoislands leads to layered biomolecular architectures, which are functional because bound DNA origami influences the number of capturing sites on the nanopatches for other proteins. The novel hybrid DNA origami-protein nanoarrays allow the fabrication of versatile research platforms for applications in biosensing, biophysics, and cell biology, and, in addition, represent an important step towards single-molecule protein arrays. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Improvement of the homogeneity of protein-imprinted polymer films by orientated immobilization of the template

    Energy Technology Data Exchange (ETDEWEB)

    Liu Lijian; Zheng Jingjing; Fang Guijie [Key Laboratory of Fermentation Engineering (Ministry of Education), College of Bioengineering, Hubei University of Technology, Nanhu Li Jia Dun 1, Wuhan 430068 (China); Xie Weihong, E-mail: weihong.xie@yahoo.com.cn [Key Laboratory of Fermentation Engineering (Ministry of Education), College of Bioengineering, Hubei University of Technology, Nanhu Li Jia Dun 1, Wuhan 430068 (China)

    2012-05-13

    Highlights: Black-Right-Pointing-Pointer MPH was genetically modified at its C-terminal with (Gly-Ser){sub 5}-Cys. Black-Right-Pointing-Pointer MPH-L was immobilized with fixed orientation via disulfide chemistry. Black-Right-Pointing-Pointer The immobilized MPH-L retained the activity of MPH. Black-Right-Pointing-Pointer MPH-L formed a homogeneous template. Black-Right-Pointing-Pointer Homogeneous MIP film was obtained with orientated immobilization of the template. - Abstract: A method for preparing homogeneous protein-imprinted polymer films with orientated immobilization of template is described. The template methyl parathion hydrolase (MPH) was modified with a peptide linker (Gly-Ser){sub 5}-Cys and was immobilized on a cover glass with a fixed orientation via the linker. The activity of the fusion enzyme (MPH-L) was evaluated by determining the product's absorbance at 405 nm (A{sub 405}). Both the free and the immobilized MPH-L showed higher retention of the bioactivity than the wide type enzyme (MPH-W) as revealed by the A{sub 405} values for MPH-L{sub free}/MPH-W{sub free} (1.159/1.111) and for MPH-L{sub immobilized}/MPH-W{sub immobilized} (0.348/0.118). The immobilized MPH-L also formed a more homogeneous template stamp compared to the immobilized MPH-W. The molecularly imprinted polymer films prepared with the immobilized MPH-L exhibited high homogeneity with low Std. Deviations of 80 and 200 from the CL intensity mean volumes which were observed for batch-prepared films and an individual film, respectively. MPH-L-imprinted polymer film also had a larger template binding capacity indicated by higher CL intensity mean volume of 3900 INT over 2500 INT for MPH-W-imprinted films. The imprinted film prepared with the orientated immobilization of template showed an imprinting factor of 1.7, while the controls did not show an imprinting effect.

  12. Integrating enzyme immobilization and protein engineering: An alternative path for the development of novel and improved industrial biocatalysts.

    Science.gov (United States)

    Bernal, Claudia; Rodríguez, Karen; Martínez, Ronny

    2018-06-09

    Enzyme immobilization often achieves reusable biocatalysts with improved operational stability and solvent resistance. However, these modifications are generally associated with a decrease in activity or detrimental modifications in catalytic properties. On the other hand, protein engineering aims to generate enzymes with increased performance at specific conditions by means of genetic manipulation, directed evolution and rational design. However, the achieved biocatalysts are generally generated as soluble enzymes, -thus not reusable- and their performance under real operational conditions is uncertain. Combined protein engineering and enzyme immobilization approaches have been employed as parallel or consecutive strategies for improving an enzyme of interest. Recent reports show efforts on simultaneously improving both enzymatic and immobilization components through genetic modification of enzymes and optimizing binding chemistry for site-specific and oriented immobilization. Nonetheless, enzyme engineering and immobilization are usually performed as separate workflows to achieve improved biocatalysts. In this review, we summarize and discuss recent research aiming to integrate enzyme immobilization and protein engineering and propose strategies to further converge protein engineering and enzyme immobilization efforts into a novel "immobilized biocatalyst engineering" research field. We believe that through the integration of both enzyme engineering and enzyme immobilization strategies, novel biocatalysts can be obtained, not only as the sum of independently improved intrinsic and operational properties of enzymes, but ultimately tailored specifically for increased performance as immobilized biocatalysts, potentially paving the way for a qualitative jump in the development of efficient, stable biocatalysts with greater real-world potential in challenging bioprocess applications. Copyright © 2018. Published by Elsevier Inc.

  13. Solid-phase synthesis of protein-polymers on reversible immobilization supports.

    Science.gov (United States)

    Murata, Hironobu; Carmali, Sheiliza; Baker, Stefanie L; Matyjaszewski, Krzysztof; Russell, Alan J

    2018-02-27

    Facile automated biomacromolecule synthesis is at the heart of blending synthetic and biologic worlds. Full access to abiotic/biotic synthetic diversity first occurred when chemistry was developed to grow nucleic acids and peptides from reversibly immobilized precursors. Protein-polymer conjugates, however, have always been synthesized in solution in multi-step, multi-day processes that couple innovative chemistry with challenging purification. Here we report the generation of protein-polymer hybrids synthesized by protein-ATRP on reversible immobilization supports (PARIS). We utilized modified agarose beads to covalently and reversibly couple to proteins in amino-specific reactions. We then modified reversibly immobilized proteins with protein-reactive ATRP initiators and, after ATRP, we released and analyzed the protein polymers. The activity and stability of PARIS-synthesized and solution-synthesized conjugates demonstrated that PARIS was an effective, rapid, and simple method to generate protein-polymer conjugates. Automation of PARIS significantly reduced synthesis/purification timelines, thereby opening a path to changing how to generate protein-polymer conjugates.

  14. Scleroglucan-borax hydrogel: a flexible tool for redox protein immobilization.

    Science.gov (United States)

    Frasconi, Marco; Rea, Sara; Matricardi, Pietro; Favero, Gabriele; Mazzei, Franco

    2009-09-15

    A highly stable biological film was prepared by casting an aqueous dispersion of protein and composite hydrogel obtained from the polysaccharide Scleroglucan (Sclg) and borax as a cross-linking agent. Heme proteins, such as hemoglobin (Hb), myoglobin (Mb), and horseradish peroxidase (HRP), were chosen as model proteins to investigate the immobilized system. A pair of well-defined quasi-reversible redox peaks, characteristics of the protein heme FeII/FeIII redox couples, were obtained at the Sclg-borax/proteins films on pyrolytic graphite (PG) electrodes, as a consequence of the direct electron transfer between the protein and the PG electrode. A full characterization of the electron transfer kinetic was performed by opportunely modeling data obtained from cyclic voltammetry and square wave voltammetry experiments. The efficiency of our cross-linking approach was investigated by studying the influence of different borax groups percentage in the Sclg matrix, revealing the versatility of this hydrogel in the immobilization of redox proteins. The native conformation of the three heme proteins entrapped in the hydrogel films were proved to be unchanged, reflected by the unaltered Soret adsorption band and by the catalytic activity toward hydrogen peroxide (H2O2). The main kinetic parameters, such as the apparent Michaelis-Menten constant, for the electrocatalytic reaction were also evaluated. The peculiar characteristics of Sclg-borax matrix make it possible to find wide opportunities as proteins immobilizing agent for studies of direct electrochemistry and biosensors development.

  15. Protein-based inverse opals: A novel support for enzyme immobilization.

    Science.gov (United States)

    Jiang, Yanjun; Sun, Wenya; Wang, Yaping; Wang, Lihui; Zhou, Liya; Gao, Jing; He, Ying; Ma, Li; Zhang, Xu

    2017-01-01

    In this study, protein-based inverse opals were prepared for the first time by using the colloidal crystal templating method. The preparation process involved three steps including filling the templates with protein molecules, crosslinking, and template removal. The obtained inverse opals were used to immobilize Penicillin G acylase (PGA) because of its intrinsic biocompatible property. The immobilization process was optimized and the properties of the immobilized PGA (PGA@IO) were investigated. PGA@IO exhibited improved thermal and pH stability compared with its free counterpart. After reusing nine times, it retained 70% of the initial activity. Besides, the PGA@IO retained high activity during the hydrolysis reactions in continuous catalysis in packed-bed reactor (PBR) after 15 days. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Immobilized sialyltransferase fused to a fungal biotin-binding protein: Production, properties, and applications.

    Science.gov (United States)

    Kajiwara, Hitomi; Tsunashima, Masako; Mine, Toshiki; Takakura, Yoshimitsu; Yamamoto, Takeshi

    2016-04-01

    A β-galactoside α2,6-sialyltransferase (ST) from the marine bacterium Photobacterium sp. JT-ISH-224 with a broad acceptor substrate specificity was fused to a fungal biotin-binding protein tamavidin 2 (TM2) to produce immobilized enzyme. Specifically, a gene for the fusion protein, in which ST from Photobacterium sp. JT-ISH-224 and TM2 were connected via a peptide linker (ST-L-TM2) was constructed and expressed in Escherichia coli. The ST-L-TM2 was produced in the soluble form with a yield of approximately 15,000 unit/300 ml of the E. coli culture. The ST-L-TM2 was partially purified and part of it was immobilized onto biotin-bearing magnetic microbeads. The immobilized ST-L-TM2 onto microbeads could be used at least seven consecutive reaction cycles with no observed decrease in enzymatic activity. In addition, the optimum pH and temperature of the immobilized enzyme were changed compared to those of a free form of the ST. Considering these results, it was strongly expected that the immobilized ST-L-TM2 was a promising tool for the production of various kind of sialoligosaccharides. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  17. Plasma treatment of paper for protein immobilization on paper-based chemiluminescence immunodevice.

    Science.gov (United States)

    Zhao, Mei; Li, Huifang; Liu, Wei; Guo, Yumei; Chu, Weiru

    2016-05-15

    A novel protein immobilization method based on plasma treatment of paper on the low-cost paper-based immunodevice was established in this work. By using a benchtop plasma cleaner, the paper microzone was treated by oxygen plasma treatment for 4 min and then the antibody can be directly immobilized on the paper surface. Aldehyde group was produced after the plasma treatment, which can be verified from the fourier transform infrared spectroscopy (FT-IR) spectra and x-ray photoelectron spectroscopy (XPS) spectra. By linked to aldehyde group, the antibody can be immobilized on the paper surface without any other pretreatment. A paper-based immunodevice was introduced here through this antibody immobilization method. With sandwich chemiluminescence (CL) immunoassay method, the paper-based immunodevice was successfully performed for carcinoembryonic antigen (CEA) detection in human serum with a linear range of 0.1-80.0 ng/mL. The detection limit was 0.03 ng/mL, which was 30 times lower than the clinical CEA level. Comparing to the other protein immobilization methods on paper-based device, this strategy was faster and simpler and had potential applications in point-of-care testing, public health and environmental monitoring. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Immobilization of ferrocene-modified SNAP-fusion proteins

    NARCIS (Netherlands)

    Wasserberg, D.; Uhlenheuer, D.; Neirynck, P.; Neirynck, Pauline; Cabanas Danés, Jordi; Schenkel, J.H.; Ravoo, B.J.; An, Q.; Huskens, Jurriaan; Milroy, L.G.; Brunsveld, Luc; Jonkheijm, Pascal

    2013-01-01

    The supramolecular assembly of proteins on surfaces has been investigated via the site-selective incorporation of a supramolecular moiety on proteins. To this end, fluorescent proteins have been site-selectively labeled with ferrocenes, as supramolecular guest moieties, via SNAP-tag technology. The

  19. Covalent immobilization of redox protein within the mesopores of transparent conducting electrodes

    Czech Academy of Sciences Publication Activity Database

    Müller, V.; Rathouský, Jiří; Fattakhova-Rohlfing, D.

    2014-01-01

    Roč. 116, JAN 2014 (2014), s. 1-8 ISSN 0013-4686 R&D Projects: GA ČR GA104/08/0435 Institutional support: RVO:61388955 Keywords : Covalent immobilization * Porous electrodes * Redox proteins Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 4.504, year: 2014

  20. From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes.

    Science.gov (United States)

    Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

    2013-01-10

    Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.

  1. From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes

    Science.gov (United States)

    Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

    2013-01-01

    Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes. PMID:23306150

  2. Recent advances in covalent, site-specific protein immobilization [version 1; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Morten Meldal

    2016-09-01

    Full Text Available The properties of biosensors, biomedical implants, and other materials based on immobilized proteins greatly depend on the method employed to couple the protein molecules to their solid support. Covalent, site-specific immobilization strategies are robust and can provide the level of control that is desired in this kind of application. Recent advances include the use of enzymes, such as sortase A, to couple proteins in a site-specific manner to materials such as microbeads, glass, and hydrogels. Also, self-labeling tags such as the SNAP-tag can be employed. Last but not least, chemical approaches based on bioorthogonal reactions, like the azide–alkyne cycloaddition, have proven to be powerful tools. The lack of comparative studies and quantitative analysis of these immobilization methods hampers the selection process of the optimal strategy for a given application. However, besides immobilization efficiency, the freedom in selecting the site of conjugation and the size of the conjugation tag and the researcher’s expertise regarding molecular biology and/or chemical techniques will be determining factors in this regard.

  3. Evaluation of protein immobilization capacity on various carbon nanotube embedded hydrogel biomaterials.

    Science.gov (United States)

    Derkus, Burak; Emregul, Kaan Cebesoy; Emregul, Emel

    2015-11-01

    This study investigates effective immobilization of proteins, an important procedure in many fields of bioengineering and medicine, using various biomaterials. Gelatin, alginate and chitosan were chosen as polymeric carriers, and applied in both their composites and nanocomposite forms in combination with carbon nanotubes (CNTs). The prepared nano/composite structures were characterized using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TG) and contact angle analysis (CA). Electrochemical impedance spectroscopy analysis revealed gelatin composites in general to exhibit better immobilization performance relative to the native gelatin which can be attributed to enhanced film morphologies of the composite structures. Moreover, superior immobilization efficiencies were obtained with the addition of carbon nanotubes, due to their conducting and surface enhancement features, especially in the gelatin-chitosan structures due to the presence of structural active groups. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Surface functionalization of superparamagnetic nanoparticles encapsulated by chitosan for protein immobilization

    International Nuclear Information System (INIS)

    Sousa, Jose Silva de

    2010-01-01

    Nanoscience and nanotechnology have opened up numerous developments of devices and systems on the nanometer scale, with new molecular organization, properties and functions. In this context, the polymeric magnetic nanoparticles are composites formed by magnetic materials with a particle size between 1 and 100 nm combined with functional polymers. They are well-known and have been widely studied because of its applications in various technology areas. Applications on the biological and medical areas include separation and immobilization of enzymes and proteins, improved techniques of magnetic resonance imaging and diagnostic systems for controlled drug delivery. In this work, proteins were immobilized on the surface of a biopolymer combined with superparamagnetic particles of magnetite. The biopolymer chitosan was used, cross-linked and functionalized with glutaraldehyde, applicable to the biological assays. Three types of magnetic composites were obtained, which were called QM1Glu, QM2NaGlu and QM3Glu. They were characterized by X-ray diffraction, scanning electron microscopy, vibrating sample magnetometry, differential scanning calorimetry, thermogravimetry and infrared spectroscopy. They were evaluated concerning the immobilization of the proteins bovine serum albumin (BSA), collagen and trypsin. The study showed that the immobilization of proteins on the biopolymer occurred in 30 min of incubation. The magnetic composite of non functionalized chitosan (QM3) was also evaluated. For trypsin, it was found that the immobilization potential of QM3 was higher than that observed for QM3Glu. After 30 days, the trypsin of the QM3-Trip and QM3Glu-Trip was still with activity. The activity and the enzyme kinetics of the QM3Glu-Trip with the substrate BApNA were demonstrated. (author)

  5. Immobilization methods for the rapid total chemical synthesis of proteins on microtiter plates.

    Science.gov (United States)

    Zitterbart, Robert; Krumrey, Michael; Seitz, Oliver

    2017-07-01

    The chemical synthesis of proteins typically involves the solid-phase peptide synthesis of unprotected peptide fragments that are stitched together in solution by native chemical ligation (NCL). The process is slow, and throughput is limited because of the need for repeated high performance liquid chromatography purification steps after both solid-phase peptide synthesis and NCL. With an aim to provide faster access to functional proteins and to accelerate the functional analysis of synthetic proteins by parallelization, we developed a method for the high performance liquid chromatography-free synthesis of proteins on the surface of microtiter plates. The method relies on solid-phase synthesis of unprotected peptide fragments, immobilization of the C-terminal fragment and on-surface NCL with an unprotected peptide thioester in crude form. Herein, we describe the development of a suitable immobilization chemistry. We compared (i) formation of nickel(II)-oligohistidine complexes, (ii) Cu-based [2 + 3] alkine-azide cycloaddition and (iii) hydrazone ligation. The comparative study identified the hydrazone ligation as most suitable. The sequence of immobilization via hydrazone ligation, on-surface NCL and radical desulfurization furnished the targeted SH3 domains in near quantitative yield. The synthetic proteins were functional as demonstrated by an on-surface fluorescence-based saturation binding analysis. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  6. Molecular printboards as general platforms for protein immobilization

    NARCIS (Netherlands)

    Ludden, M.J.W.

    2007-01-01

    This thesis describes the selective attachment of proteins to β-cyclodextrin (βCD) self-assembled monolayers (SAMs), termed molecular printboards through multivalent orthogonal interactions. It is shown that the molecular printboards allow different assembly pathways for the build-up of (complex)

  7. Effects of immobilization and aerobic training on proteins related to intramuscular substrate storage and metabolism in young and older men

    DEFF Research Database (Denmark)

    Vigelsø Hansen, Andreas; Gram, Martin; Wiuff, Caroline

    2016-01-01

    by aerobic training in young and older men. METHODS: 17 young (23 ± 1 years, 24 ± 1 kg m(-2), and 20 ± 2% body fat) and 15 older men (68 ± 1 years; 27 ± 1 kg m(-2), and 29 ± 2% body fat) underwent 2 weeks' one leg immobilization followed by 6 weeks' cycle training. Biopsies were obtained from m. vastus...... lateralis just before immobilization (at inclusion), after immobilization, and the after 6 weeks' training. The biopsies were analyzed for muscle substrates; muscle perilipin protein (PLIN), glycogen synthase (GS), synaptosomal-associated protein of 23 kDa (SNAP23) protein content, and muscle 3-hydroxyacyl...... GS (74%) protein compared to the older men. Immobilization decreased and training restored HAD activity, GS and SNAP23 protein content in young and older men. CONCLUSION: Evidence of age-related metabolic inflexibility is presented, seen as body fat and IMTG accumulation. The question arises...

  8. Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

    Science.gov (United States)

    Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

    2016-02-01

    A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. In vivo immobilization of fusion proteins on bioplastics by the novel tag BioF.

    Science.gov (United States)

    Moldes, Cristina; García, Pedro; García, José L; Prieto, María A

    2004-06-01

    A new protein immobilization and purification system has been developed based on the use of polyhydroxyalkanoates (PHAs, or bioplastics), which are biodegradable polymers accumulated as reserve granules in the cytoplasm of certain bacteria. The N-terminal domain of the PhaF phasin (a PHA-granule-associated protein) from Pseudomonas putida GPo1 was used as a polypeptide tag (BioF) to anchor fusion proteins to PHAs. This tag provides a novel way to immobilize proteins in vivo by using bioplastics as supports. The granules carrying the BioF fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble BioF fusion protein can be obtained by a mild detergent treatment of the granule. The efficiency of this system has been demonstrated by constructing two BioF fusion products, including a functional BioF-beta-galactosidase. This is the first example of an active bioplastic consisting of a biodegradable matrix carrying an active enzyme.

  10. Micrometer sized immobilization of protein molecules onto quartz, silicium and gold.

    Science.gov (United States)

    Petersen, Steffen B.; Neves-Petersen, Maria Teresa; Klitgaard, Søren; Duroux, Meg Crookshanks

    2006-02-01

    We demonstrate that ultraviolet light can be used to make sterically oriented covalent immobilization of a large variety of protein molecules onto either gold or thiolated quartz or silicium. The reaction mechanism behind the reported new technology involves light induced breakage of disulphide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol reactive surfaces. The protein molecules in general retain their function. The size of the immobilization spot is determined by the dimension of the UV beam. In principle, the spot size may be as small as 1 micrometer or less. We have developed the necessary technology for preparing large protein arrays of enzymes and fragments of monoclonal antibodies. Dedicated Image Processing Software has been developed for making quality assessment of the protein arrays. A multitude of important application areas such as drug carriers and drug delivery, bioelectronics, carbon nanotubes, nanoparticles as well as protein glue are discussed.

  11. Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success

    International Nuclear Information System (INIS)

    Choi, Ryan; Kelley, Angela; Leibly, David; Nakazawa Hewitt, Stephen; Napuli, Alberto; Van Voorhis, Wesley

    2011-01-01

    An overview of the methods used for high-throughput cloning and protein-expression screening of SSGCID hexahistidine recombinant proteins is provided. It is demonstrated that screening for recombinant proteins that are highly recoverable from immobilized metal-affinity chromatography improves the likelihood that a protein will produce a structure. The recombinant expression of soluble proteins in Escherichia coli continues to be a major bottleneck in structural genomics. The establishment of reliable protocols for the performance of small-scale expression and solubility testing is an essential component of structural genomic pipelines. The SSGCID Protein Production Group at the University of Washington (UW-PPG) has developed a high-throughput screening (HTS) protocol for the measurement of protein recovery from immobilized metal-affinity chromatography (IMAC) which predicts successful purification of hexahistidine-tagged proteins. The protocol is based on manual transfer of samples using multichannel pipettors and 96-well plates and does not depend on the use of robotic platforms. This protocol has been applied to evaluate the expression and solubility of more than 4000 proteins expressed in E. coli. The UW-PPG also screens large-scale preparations for recovery from IMAC prior to purification. Analysis of these results show that our low-cost non-automated approach is a reliable method for the HTS demands typical of large structural genomic projects. This paper provides a detailed description of these protocols and statistical analysis of the SSGCID screening results. The results demonstrate that screening for proteins that yield high recovery after IMAC, both after small-scale and large-scale expression, improves the selection of proteins that can be successfully purified and will yield a crystal structure

  12. New tool for spreading proteins to the environment: Cry1Ab toxin immobilized to bioplastics.

    Science.gov (United States)

    Moldes, Cristina; Farinós, Gema P; de Eugenio, Laura I; García, Pedro; García, José L; Ortego, Félix; Hernández-Crespo, Pedro; Castañera, Pedro; Prieto, María A

    2006-08-01

    A new tool to provide an environmentally friendly way to deliver active proteins to the environment has been developed, based on the use of polyhydroxyalkanoate (PHA, bioplastic) granules. To illustrate this novel approach, a derived Cry1Ab insect-specific toxin protein was in vivo immobilized into PHA granules through the polypeptide tag BioF. The new toxin, named Fk-Bt1, was shown to be active against Sesamia nonagrioides (Lepidoptera: Noctuidae). The dose-mortality responses of the new toxin granule formulation (PFk-Bt1) and purified Cry1Ab have been compared, demonstrating the effectiveness of PFk-Bt1 and suggesting a common mode of action.

  13. Comparison of Zirconium Phosphonate-Modified Surfaces for Immobilizing Phosphopeptides and Phosphate-Tagged Proteins.

    Science.gov (United States)

    Forato, Florian; Liu, Hao; Benoit, Roland; Fayon, Franck; Charlier, Cathy; Fateh, Amina; Defontaine, Alain; Tellier, Charles; Talham, Daniel R; Queffélec, Clémence; Bujoli, Bruno

    2016-06-07

    Different routes for preparing zirconium phosphonate-modified surfaces for immobilizing biomolecular probes are compared. Two chemical-modification approaches were explored to form self-assembled monolayers on commercially available primary amine-functionalized slides, and the resulting surfaces were compared to well-characterized zirconium phosphonate monolayer-modified supports prepared using Langmuir-Blodgett methods. When using POCl3 as the amine phosphorylating agent followed by treatment with zirconyl chloride, the result was not a zirconium-phosphonate monolayer, as commonly assumed in the literature, but rather the process gives adsorbed zirconium oxide/hydroxide species and to a lower extent adsorbed zirconium phosphate and/or phosphonate. Reactions giving rise to these products were modeled in homogeneous-phase studies. Nevertheless, each of the three modified surfaces effectively immobilized phosphopeptides and phosphopeptide tags fused to an affinity protein. Unexpectedly, the zirconium oxide/hydroxide modified surface, formed by treating the amine-coated slides with POCl3/Zr(4+), afforded better immobilization of the peptides and proteins and efficient capture of their targets.

  14. Monte Carlo simulations of protein micropatterning in biomembranes: effects of immobile sticky obstacles

    International Nuclear Information System (INIS)

    Arnold, Andreas M; Sevcsik, Eva; Schütz, Gerhard J

    2016-01-01

    Single molecule trajectories of lipids and proteins can yield valuable information about the nanoscopic organization of the plasma membrane itself. The interpretation of such trajectories, however, is complicated, as the mobility of molecules can be affected by the presence of immobile obstacles, and the transient binding of the tracers to these obstacles. We have previously developed a micropatterning approach that allows for immobilizing a plasma membrane protein and probing the diffusional behavior of a putative interaction partner in living cells. Here, we provide guidelines on how this micropatterning approach can be extended to quantify interaction parameters between plasma membrane constituents in their natural environment. We simulated a patterned membrane system and evaluated the effect of different surface densities of patterned immobile obstacles on the relative mobility as well as the surface density of diffusing tracers. In the case of inert obstacles, the size of the obstacle can be assessed from its surface density at the percolation threshold, which in turn can be extracted from the diffusion behavior of the tracer. For sticky obstacles, 2D dissociation constants can be determined from the tracer diffusion or surface density. (paper)

  15. Sonochemical synthesis of (3-aminopropyl)triethoxysilane-modified monodispersed silica nanoparticles for protein immobilization

    International Nuclear Information System (INIS)

    Shen, Shou-Cang; Ng, Wai Kiong; Chia, Leonard; Dong, Yuan-Cai; Tan, Reginald B.H.

    2011-01-01

    Graphical abstract: 3-Aminopropyltriethoxysilane modified monodispersed silica nanoparticles were synthesized by rapid sonochemical co-condensation to achieve high capability for protein immobilization. Highlights: → Amino-modified monodispersed silica nanoparticles were synthesized by rapid co-condensation. → Strong positive charge was created by aminopropyl-modification. → Capability for immobilization of negatively charged protein was enhanced. → Electrostatic interaction between proteins and surface contributed to the enhanced adsorption. -- Abstract: 3-Aminopropyltriethoxysilane modified monodispersed silica nanoparticles were synthesized by a rapid sonochemical co-condensation synthesis procedure. The chemical nature of surface organic modifier on the obtained modified silica nanoparticle was characterized by 13 C and 29 Si MAS Nuclear Magnetic Resonance (NMR) spectroscopies, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA)- differential scanning calorimetry (DSC). Due to the strengthened positive surface charge of the silica nanoparticles by the modification with aminopropyl groups, the capability for bovine serum albumin (BSA) adsorption was significantly increased as compared with bare silica nanoparticles. 80 mg/g BSA was adsorbed on modified silica nanoparticles, whereas only 20 mg/g BSA could be loaded on pure silica nanoparticles. The enhanced positive surface charge repelled proteins with net positive charge and the modified silica nanoparticles exhibited negligible adsorption of lysozyme, thus a selective adsorption of proteins could be achieved.

  16. Protein immobilization on epoxy-activated thin polymer films: effect of surface wettability and enzyme loading.

    Science.gov (United States)

    Chen, Bo; Pernodet, Nadine; Rafailovich, Miriam H; Bakhtina, Asya; Gross, Richard A

    2008-12-02

    A series of epoxy-activated polymer films composed of poly(glycidyl methacrylate/butyl methacrylate/hydroxyethyl methacrylate) were prepared. Variation in comonomer composition allowed exploration of relationships between surface wettability and Candida antartica lipase B (CALB) binding to surfaces. By changing solvents and polymer concentrations, suitable conditions were developed for preparation by spin-coating of uniform thin films. Film roughness determined by AFM after incubation in PBS buffer for 2 days was less than 1 nm. The occurrence of single CALB molecules and CALB aggregates at surfaces was determined by AFM imaging and measurements of volume. Absolute numbers of protein monomers and multimers at surfaces were used to determine values of CALB specific activity. Increased film wettability, as the water contact angle of films increased from 420 to 550, resulted in a decreased total number of immobilized CALB molecules. With further increases in the water contact angle of films from 55 degrees to 63 degrees, there was an increased tendency of CALB molecules to form aggregates on surfaces. On all flat surfaces, two height populations, differing by more than 30%, were observed from height distribution curves. They are attributed to changes in protein conformation and/or orientation caused by protein-surface and protein-protein interactions. The fraction of molecules in these populations changed as a function of film water contact angle. The enzyme activity of immobilized films was determined by measuring CALB-catalyzed hydrolysis of p-nitrophenyl butyrate. Total enzyme specific activity decreased by decreasing film hydrophobicity.

  17. Sonochemical synthesis of (3-aminopropyl)triethoxysilane-modified monodispersed silica nanoparticles for protein immobilization

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Shou-Cang, E-mail: shen_shoucang@ices.a-star.edu.sg [Institute of Chemical and Engineering Sciences, A-STAR (Agency for Science, Technology and Research), 1 Pesek Road, Jurong Island, Singapore 627833 (Singapore); Ng, Wai Kiong; Chia, Leonard; Dong, Yuan-Cai [Institute of Chemical and Engineering Sciences, A-STAR (Agency for Science, Technology and Research), 1 Pesek Road, Jurong Island, Singapore 627833 (Singapore); Tan, Reginald B.H., E-mail: reginald_tan@ices.a-star.edu.sg [Institute of Chemical and Engineering Sciences, A-STAR (Agency for Science, Technology and Research), 1 Pesek Road, Jurong Island, Singapore 627833 (Singapore); Department of Chemical and Biomolecular Engineering, The National University of Singapore, 4 Engineering Drive 4, Singapore 117576 (Singapore)

    2011-10-15

    Graphical abstract: 3-Aminopropyltriethoxysilane modified monodispersed silica nanoparticles were synthesized by rapid sonochemical co-condensation to achieve high capability for protein immobilization. Highlights: {yields} Amino-modified monodispersed silica nanoparticles were synthesized by rapid co-condensation. {yields} Strong positive charge was created by aminopropyl-modification. {yields} Capability for immobilization of negatively charged protein was enhanced. {yields} Electrostatic interaction between proteins and surface contributed to the enhanced adsorption. -- Abstract: 3-Aminopropyltriethoxysilane modified monodispersed silica nanoparticles were synthesized by a rapid sonochemical co-condensation synthesis procedure. The chemical nature of surface organic modifier on the obtained modified silica nanoparticle was characterized by {sup 13}C and {sup 29}Si MAS Nuclear Magnetic Resonance (NMR) spectroscopies, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA)- differential scanning calorimetry (DSC). Due to the strengthened positive surface charge of the silica nanoparticles by the modification with aminopropyl groups, the capability for bovine serum albumin (BSA) adsorption was significantly increased as compared with bare silica nanoparticles. 80 mg/g BSA was adsorbed on modified silica nanoparticles, whereas only 20 mg/g BSA could be loaded on pure silica nanoparticles. The enhanced positive surface charge repelled proteins with net positive charge and the modified silica nanoparticles exhibited negligible adsorption of lysozyme, thus a selective adsorption of proteins could be achieved.

  18. Protein immobilization on Ni(II) ion patterns prepared by microcontact printing and dip-pen nanolithography

    NARCIS (Netherlands)

    Wu, Chien-Ching; Reinhoudt, David N; Otto, Cees; Velders, Aldrik H; Subramaniam, Vinod

    2010-01-01

    An indirect method of protein patterning by using Ni(II) ion templates for immobilization via a specific metal-protein interaction is described. A nitrilotriacetic acid (NTA)-terminated self-assembled monolayer (SAM) allows oriented binding of histidine-tagged proteins via complexation with late

  19. Multi-scale carbon micro/nanofibers-based adsorbents for protein immobilization

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Shiv; Singh, Abhinav [Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India); Bais, Vaibhav Sushil Singh; Prakash, Balaji [Department of Biological Science and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India); Verma, Nishith, E-mail: nishith@iitk.ac.in [Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India); Center for Environmental Science and Engineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India)

    2014-05-01

    In the present study, different proteins, namely, bovine serum albumin (BSA), glucose oxidase (GOx) and the laboratory purified YqeH were immobilized in the phenolic resin precursor-based multi-scale web of activated carbon microfibers (ACFs) and carbon nanofibers (CNFs). These biomolecules are characteristically different from each other, having different structure, number of parent amino acid molecules and isoelectric point. CNF was grown on ACF substrate by chemical vapor deposition, using Ni nanoparticles (Nps) as the catalyst. The ultra-sonication of the CNFs was carried out in acidic medium to remove Ni Nps from the tip of the CNFs to provide additional active sites for adsorption. The prepared material was directly used as an adsorbent for proteins, without requiring any additional treatment. Several analytical techniques were used to characterize the prepared materials, including scanning electron microscopy, Fourier transform infrared spectroscopy, BET surface area, pore-size distribution, and UV–vis spectroscopy. The adsorption capacities of prepared ACFs/CNFs in this study were determined to be approximately 191, 39 and 70 mg/g for BSA, GOx and YqeH, respectively, revealing that the carbon micro-nanofibers forming synthesized multi-scale web are efficient materials for the immobilization of protein molecules. - Highlights: • Ni metal Np-dispersed carbon micro-nanofibers (ACFs/CNFs) are prepared. • ACFs/CNFs are mesoporous. • Significant adsorption of BSA, GOx and YqeH is observed on ACFs/CNFs. • Multi-scale web of ACFs/CNFs is effective for protein immobilization.

  20. Effects of immobilization and aerobic training on proteins related to intramuscular substrate storage and metabolism in young and older men.

    Science.gov (United States)

    Vigelsø, Andreas; Gram, Martin; Wiuff, Caroline; Hansen, Christina Neigaard; Prats, Clara; Dela, Flemming; Helge, Jørn Wulff

    2016-03-01

    Aging and inactivity lead to skeletal muscle metabolic inflexibility, but the underlying molecular mechanisms are not entirely elucidated. Therefore, we investigated how muscle lipid and glycogen stores and major regulatory proteins were affected by short-term immobilization followed by aerobic training in young and older men. 17 young (23 ± 1 years, 24 ± 1 kg m(-2), and 20 ± 2% body fat) and 15 older men (68 ± 1 years; 27 ± 1 kg m(-2), and 29 ± 2% body fat) underwent 2 weeks' one leg immobilization followed by 6 weeks' cycle training. Biopsies were obtained from m. vastus lateralis just before immobilization (at inclusion), after immobilization, and the after 6 weeks' training. The biopsies were analyzed for muscle substrates; muscle perilipin protein (PLIN), glycogen synthase (GS), synaptosomal-associated protein of 23 kDa (SNAP23) protein content, and muscle 3-hydroxyacyl-CoA dehydrogenase (HAD) activity The older men had higher intramuscular triglyceride (IMTG) (73 %) and Glycogen (16%) levels compared to the young men, and IMTG tended to increase with immobilization. PLIN2 and 3 protein content increased with immobilization in the older men only. The young men had higher GS (74%) protein compared to the older men. Immobilization decreased and training restored HAD activity, GS and SNAP23 protein content in young and older men. Evidence of age-related metabolic inflexibility is presented, seen as body fat and IMTG accumulation. The question arises as to whether IMTG accumulation in the older men is caused by or leading to the increase in PLIN2 and 3 protein content. Training decreased body fat and IMTG levels in both young and older men; hence, training should be prioritized to reduce the detrimental effect of aging on metabolism.

  1. Fabrication of protein microarrays for alpha fetoprotein detection by using a rapid photo-immobilization process

    Directory of Open Access Journals (Sweden)

    Sirasa Yodmongkol

    2016-03-01

    Full Text Available In this study, protein microarrays based on sandwich immunoassays are generated to quantify the amount of alpha fetoprotein (AFP in blood serum. For chip generation a mixture of capture antibody and a photoactive copolymer consisting of N,N-dimethylacrylamide (DMAA, methacryloyloxy benzophenone (MaBP, and Na-4-styrenesulfonate (SSNa was spotted onto unmodified polymethyl methacrylate (PMMA substrates. Subsequently to printing of the microarray, the polymer and protein were photochemically cross-linked and the forming, biofunctionalized hydrogels simultaneously bound to the chip surface by short UV- irradiation. The obtained biochip was incubated with AFP antigen, followed by biotinylated AFP antibody and streptavidin-Cy5 and the fluorescence signal read-out. The developed microarray biochip covers the range of AFP in serum samples such as maternal serum in the range of 5 and 100 ng/ml. The chip production process is based on a fast and simple immobilization process, which can be applied to conventional plastic surfaces. Therefore, this protein microarray production process is a promising method to fabricate biochips for AFP screening processes. Keywords: Photo-immobilization, Protein microarray, Alpha fetoprotein, Hydrogel, 3D surface, Down syndrome

  2. Nickel electrodes as a cheap and versatile platform for studying structure and function of immobilized redox proteins

    Energy Technology Data Exchange (ETDEWEB)

    Han, Xiao Xia [State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Street, Changchun 130012 (China); Institut für Chemie, Technische Universität Berlin, Sekr. PC14, Strasse des 17. Juni 135, D-10623 Berlin (Germany); Li, Junbo [State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Street, Changchun 130012 (China); Öner, Ibrahim Halil [Institut für Chemie, Technische Universität Berlin, Sekr. PC14, Strasse des 17. Juni 135, D-10623 Berlin (Germany); Zhao, Bing [State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Street, Changchun 130012 (China); Leimkühler, Silke [Institut für Biochemie und Biologie, Universität Potsdam, Karl-Liebknecht Straße 24-25, H. 25, Golm D-14476 (Germany); Hildebrandt, Peter [Institut für Chemie, Technische Universität Berlin, Sekr. PC14, Strasse des 17. Juni 135, D-10623 Berlin (Germany); Weidinger, Inez M., E-mail: i.weidinger@mailbox.tu-berlin.de [Institut für Chemie, Technische Universität Berlin, Sekr. PC14, Strasse des 17. Juni 135, D-10623 Berlin (Germany)

    2016-10-19

    Practical use of many bioelectronic and bioanalytical devices is limited by the need of expensive materials and time consuming fabrication. Here we demonstrate the use of nickel electrodes as a simple and cheap solid support material for bioelectronic applications. The naturally nanostructured electrodes showed a surprisingly high electromagnetic surface enhancement upon light illumination such that immobilization and electron transfer reactions of the model redox proteins cytochrome b{sub 5} (Cyt b{sub 5}) and cytochrome c (Cyt c) could be followed via surface enhanced resonance Raman spectroscopy. It could be shown that the nickel surface, when used as received, promotes a very efficient binding of the proteins upon preservation of their native structure. The immobilized redox proteins could efficiently exchange electrons with the electrode and could even act as an electron relay between the electrode and solubilized myoglobin. Our results open up new possibility for nickel electrodes as an exceptional good support for bioelectronic devices and biosensors on the one hand and for surface enhanced spectroscopic investigations on the other hand. - Highlights: • Nickel electrodes were used without further functionalization as supports for various redox proteins. • It was possible to monitor the immobilized proteins via surface enhanced Raman spectroscopy. • The native structure of the immobilized proteins was preserved and they could exchange electrons with the Ni electrode. • The immobilized redox proteins worked as an electron relay between electrode and solubilized myoglobin.

  3. Protein immobilization on the surface of liposomes via carbodiimide activation in the presence of N-hydroxysulfosuccinimide.

    Science.gov (United States)

    Bogdanov, A A; Klibanov, A L; Torchilin, V P

    1988-04-25

    A method of the covalent immobilization of proteins on the surface of liposomes, containing 10% (by mol) of N-glutaryl phosphatidylethanolamine, is described. Carboxylic groups of liposomal N-glutaryl phosphatidylethanolamine were activated in the presence of water-soluble carbodiimide and N-hydroxysulfosuccinimide and reacted subsequently with protein amino groups. The liposome-protein conjugates formed contained up to 5 x 10(-4) mol protein/mol lipid. Lectins (RCA1 and WGA) upon immobilization on liposomes retained saccharide specificity and the ability to agglutinate red blood cells. The immobilization of mouse monoclonal IgG in a ratio of 3.5 x 10(-4) mol IgG/mol lipid was achieved. The liposome activation in the absence of N-hydroxysulfosuccinimide resulted in a 2-fold decrease of protein coupling yields.

  4. Immobilization of small molecules and proteins by radio-derivatized polystyrene

    International Nuclear Information System (INIS)

    Varga, J.M.; Fritsch, P.

    1990-01-01

    When molded polystyrene (PS) products (e.g., microtiter plates) or latex particles are irradiated with high-energy (1-10 Mrads) gamma rays in the presence of nonpolymerizable small molecules such as aromatic amines, some of these molecules incorporate into PS, which leads to the formation of radio-derivatized PS (RDPS). Two classes of RDPS can be identified regarding their ability for immobilization of biologically important molecules: (1) reactive RDPS that are able to form covalent bonds with molecules such as proteins without the help of cross-linkers, and (2) functionalized RDPS that can be used for the immobilization of molecules with activators (e.g., carbodiimides) or cross-linkers. The method can be used for the production of low-noise supports for binding assays. Most of the RDPS can be produced without impairment of the optical quality of PS, making derivatized microtiter plates suitable for colorimetric assays. The principle can be applied for the preparation of affinity sorbents, e.g., for high-performance affinity chromatography and for the immobilization of enzymes using latex PS particles

  5. The amino-terminal structure of human fragile X mental retardation protein obtained using precipitant-immobilized imprinted polymers

    Science.gov (United States)

    Hu, Yufeng; Chen, Zhenhang; Fu, Yanjun; He, Qingzhong; Jiang, Lun; Zheng, Jiangge; Gao, Yina; Mei, Pinchao; Chen, Zhongzhou; Ren, Xueqin

    2015-03-01

    Flexibility is an intrinsic property of proteins and essential for their biological functions. However, because of structural flexibility, obtaining high-quality crystals of proteins with heterogeneous conformations remain challenging. Here, we show a novel approach to immobilize traditional precipitants onto molecularly imprinted polymers (MIPs) to facilitate protein crystallization, especially for flexible proteins. By applying this method, high-quality crystals of the flexible N-terminus of human fragile X mental retardation protein are obtained, whose absence causes the most common inherited mental retardation. A novel KH domain and an intermolecular disulfide bond are discovered, and several types of dimers are found in solution, thus providing insights into the function of this protein. Furthermore, the precipitant-immobilized MIPs (piMIPs) successfully facilitate flexible protein crystal formation for five model proteins with increased diffraction resolution. This highlights the potential of piMIPs for the crystallization of flexible proteins.

  6. One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

    DEFF Research Database (Denmark)

    Lee, Yu-Chen; Block, Gregory; Chen, Huiwen

    2008-01-01

    We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane...... proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment...... of plasma membrane marker 5'-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5'-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1...

  7. Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates.

    Directory of Open Access Journals (Sweden)

    Christy Catherine

    Full Text Available Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA.

  8. Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases

    International Nuclear Information System (INIS)

    Besanger, Travis R.; Hodgson, Richard J.; Green, James R.A.; Brennan, John D.

    2006-01-01

    Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol-gel processing route. While such columns proved to be useful for applications such as screening of protein-ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low μL/min range. Using the enzyme γ-glutamyl transpeptidase (γ-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns with different silica morphologies as a function of flowrate and backpressure using the enzyme reactor assay mode. It was found that the apparent activity of the enzyme was highest in mesoporous columns that retained high levels of enzyme. In such columns, enzyme activity increased by ∼2-fold with increases in both flowrate (from 250 to 1000 nL/min) and backpressure generated (from 500 to 2100 psi) during the chromatographic activity assay owing to increases in k cat and decreases in K M , switching from diffusion controlled to reaction controlled conditions at ca. 2000 psi. These results suggest that columns with minimal macropore volumes (<5%) are advantageous for the entrapment of soluble proteins for bioaffinity and bioreactor chromatography

  9. Synthesis of an oligonucleotide-derivatized amphipol and its use to trap and immobilize membrane proteins

    DEFF Research Database (Denmark)

    Bon, Christel Le; Della Pia, Eduardo Antonio; Giusti, Fabrice

    2014-01-01

    'OligAPol' have been investigated. Grafting was performed by reacting an ODN carrying an amine-terminated arm with the carboxylates of A8-35. The use of OligAPol for trapping MPs and immobilizing them onto solid supports was tested using bacteriorhodopsin (BR) and the transmembrane domain of Escherichia...... coli outer membrane protein A (tOmpA) as model proteins. BR and OligAPol form water-soluble complexes in which BR remains in its native conformation. Hybridization of the ODN arm with a complementary ODN was not hindered by the assembly of OligAPol into particles, nor by its association with BR. BR....../OligAPol and tOmpA/OligAPol complexes could be immobilized onto either magnetic beads or gold nanoparticles grafted with the complementary ODN, as shown by spectroscopic measurements, fluorescence microscopy and the binding of anti-BR and anti-tOmpA antibodies. OligAPols provide a novel, highly versatile...

  10. The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice

    Science.gov (United States)

    You, Jae-Sung; Anderson, Garrett B.; Dooley, Matthew S.; Hornberger, Troy A.

    2015-01-01

    ABSTRACT The maintenance of skeletal muscle mass contributes substantially to health and to issues associated with the quality of life. It has been well recognized that skeletal muscle mass is regulated by mechanically induced changes in protein synthesis, and that signaling by mTOR is necessary for an increase in protein synthesis and the hypertrophy that occurs in response to increased mechanical loading. However, the role of mTOR signaling in the regulation of protein synthesis and muscle mass during decreased mechanical loading remains largely undefined. In order to define the role of mTOR signaling, we employed a mouse model of hindlimb immobilization along with pharmacological, mechanical and genetic means to modulate mTOR signaling. The results first showed that immobilization induced a decrease in the global rates of protein synthesis and muscle mass. Interestingly, immobilization also induced an increase in mTOR signaling, eIF4F complex formation and cap-dependent translation. Blocking mTOR signaling during immobilization with rapamycin not only impaired the increase in eIF4F complex formation, but also augmented the decreases in global protein synthesis and muscle mass. On the other hand, stimulating immobilized muscles with isometric contractions enhanced mTOR signaling and rescued the immobilization-induced decrease in global protein synthesis through a rapamycin-sensitive mechanism that was independent of ribosome biogenesis. Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation. Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size. Therefore, we conclude that the activation of mTOR signaling is both necessary and sufficient to alleviate the decreases in protein synthesis and muscle mass that occur during immobilization. Furthermore, these results indicate

  11. Specific and reversible immobilization of proteins tagged to the affinity polypeptide C-LytA on functionalized graphite electrodes.

    Directory of Open Access Journals (Sweden)

    Daniel Bello-Gil

    Full Text Available We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.

  12. Specific and Reversible Immobilization of Proteins Tagged to the Affinity Polypeptide C-LytA on Functionalized Graphite Electrodes

    Science.gov (United States)

    Bello-Gil, Daniel; Maestro, Beatriz; Fonseca, Jennifer; Feliu, Juan M.; Climent, Víctor; Sanz, Jesús M.

    2014-01-01

    We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field. PMID:24498237

  13. Trypsin immobilization in ordered porous polymer membranes for effective protein digestion

    International Nuclear Information System (INIS)

    Qiao, Juan; Kim, Jin Yong; Wang, Yuan Yuan; Qi, Li; Wang, Fu Yi; Moon, Myeong Hee

    2016-01-01

    Fast and effective protein digestion is a vital process for mass spectrometry (MS) based protein analysis. This study introduces a porous polymer membrane enzyme reactor (PPMER) coupled to nanoflow liquid chromatography-tandem MS (nLC-ESI-MS/MS) for on-line digestion and analysis of proteins. Poly (styrene-co-maleic anhydride) (PS-co-MAn) was fabricated by the breath figure method to make a porous polymer membrane in which the MAn group was covalently bound to enzyme. Based on this strategy, microscale PPMER (μPPMER) was constructed for on-line connection with the nLC-ESI-MS/MS system. Its capability for enzymatic digestion with bovine serum albumin (BSA) was evaluated with varied digestion periods. The on-line proteolysis of BSA and subsequent analysis with μPPMER-nLC-ESI-MS/MS revealed that peptide sequence coverage increased from 10.3% (digestion time 10 min) to 89.1% (digestion time 30 min). μPPMER can efficiently digest proteins due to the microscopic confinement effect, showing its potential application in fast protein identification and protease immobilization. Applications of on-line digestion using μPPMER with human plasma and urinary proteome samples showed that the developed on-line method yielded equivalent or better performance in protein coverage and identified more membrane proteins than the in-solution method. This may be due to easy accommodation of hydrophobic membrane proteins within membrane pores. - Highlights: • A porous polymer membrane enzyme reactor was developed. • Breath figure method was used for the fabrication of porous polymer membrane. • The enzyme reactor was coupled to nLC-ESI-MS/MS for proteins on-line digestion.

  14. Trypsin immobilization in ordered porous polymer membranes for effective protein digestion

    Energy Technology Data Exchange (ETDEWEB)

    Qiao, Juan [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190 (China); Kim, Jin Yong [Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seoul 120-749 (Korea, Republic of); Wang, Yuan Yuan [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190 (China); Qi, Li, E-mail: qili@iccas.ac.cn [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190 (China); Wang, Fu Yi [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190 (China); Moon, Myeong Hee, E-mail: mhmoon@yonsei.ac.kr [Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seoul 120-749 (Korea, Republic of)

    2016-02-04

    Fast and effective protein digestion is a vital process for mass spectrometry (MS) based protein analysis. This study introduces a porous polymer membrane enzyme reactor (PPMER) coupled to nanoflow liquid chromatography-tandem MS (nLC-ESI-MS/MS) for on-line digestion and analysis of proteins. Poly (styrene-co-maleic anhydride) (PS-co-MAn) was fabricated by the breath figure method to make a porous polymer membrane in which the MAn group was covalently bound to enzyme. Based on this strategy, microscale PPMER (μPPMER) was constructed for on-line connection with the nLC-ESI-MS/MS system. Its capability for enzymatic digestion with bovine serum albumin (BSA) was evaluated with varied digestion periods. The on-line proteolysis of BSA and subsequent analysis with μPPMER-nLC-ESI-MS/MS revealed that peptide sequence coverage increased from 10.3% (digestion time 10 min) to 89.1% (digestion time 30 min). μPPMER can efficiently digest proteins due to the microscopic confinement effect, showing its potential application in fast protein identification and protease immobilization. Applications of on-line digestion using μPPMER with human plasma and urinary proteome samples showed that the developed on-line method yielded equivalent or better performance in protein coverage and identified more membrane proteins than the in-solution method. This may be due to easy accommodation of hydrophobic membrane proteins within membrane pores. - Highlights: • A porous polymer membrane enzyme reactor was developed. • Breath figure method was used for the fabrication of porous polymer membrane. • The enzyme reactor was coupled to nLC-ESI-MS/MS for proteins on-line digestion.

  15. Mechanical desorption of immobilized proteins using carbon dioxide aerosols for reusable biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Renu; Hong, Seongkyeol [School of Mechanical and Nuclear Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of); Jang, Jaesung, E-mail: jjang@unist.ac.kr [School of Mechanical and Nuclear Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of); Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of); School of Materials Science and Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of)

    2015-01-01

    Highlights: • Immobilized proteins were removed using carbon dioxide aerosols. • We observed high removal efficiencies due to the aerosol treatment. • We confirmed the removal with FTIR and X-ray photoelectron spectroscopy. • This CO{sub 2} aerosol treatment did not undermine re-functionalization. • This technique is a fast and damage-free method to reuse a sensor surface. - Abstract: Reusability of a biosensor has recently received considerable attention, and it is closely related with the effective desorption of probe molecules. We present a novel mechanical desorption technique to reuse biosensors by using periodic jets of carbon dioxide (CO{sub 2}) aerosols (a mixture of solid and gaseous CO{sub 2}), and demonstrate its feasibility by removing physically adsorbed and covalently bonded fluorescent proteins i.e., Escherichia coli fluorescein isothiocyanate antibody and bovine serum albumin (E. coli FITC–Ab and FITC–BSA) from silicon chips. The proteins on the chip surfaces were measured by fluorescent images before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured for various concentrations (1–20 μg mL{sup −1}) of E. coli FITC–Ab and FITC–BSA with two different removal cycles (5 and 11 cycles; each cycle: 8 s). We observed high removal efficiencies (>93.5% for physically adsorbed Ab and >84.6% for covalently bonded Ab) at 11 cycle aerosol treatment. This CO{sub 2} aerosol treatment did not undermine re-functionalization, which was confirmed by the fluorescent images of FITC–Abs for fresh and reused chips. Desorption of the immobilized layers was validated by Fourier transform infrared and X-ray photoelectron spectroscopic analyses. We also conducted an experiment on the regeneration of E. coli sensing chips using this aerosol treatment, and the chips were re-used 5 times successfully. This mechanical desorption technique is a highly effective and novel strategy for reusable biosensors.

  16. Mechanical desorption of immobilized proteins using carbon dioxide aerosols for reusable biosensors

    International Nuclear Information System (INIS)

    Singh, Renu; Hong, Seongkyeol; Jang, Jaesung

    2015-01-01

    Highlights: • Immobilized proteins were removed using carbon dioxide aerosols. • We observed high removal efficiencies due to the aerosol treatment. • We confirmed the removal with FTIR and X-ray photoelectron spectroscopy. • This CO 2 aerosol treatment did not undermine re-functionalization. • This technique is a fast and damage-free method to reuse a sensor surface. - Abstract: Reusability of a biosensor has recently received considerable attention, and it is closely related with the effective desorption of probe molecules. We present a novel mechanical desorption technique to reuse biosensors by using periodic jets of carbon dioxide (CO 2 ) aerosols (a mixture of solid and gaseous CO 2 ), and demonstrate its feasibility by removing physically adsorbed and covalently bonded fluorescent proteins i.e., Escherichia coli fluorescein isothiocyanate antibody and bovine serum albumin (E. coli FITC–Ab and FITC–BSA) from silicon chips. The proteins on the chip surfaces were measured by fluorescent images before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured for various concentrations (1–20 μg mL −1 ) of E. coli FITC–Ab and FITC–BSA with two different removal cycles (5 and 11 cycles; each cycle: 8 s). We observed high removal efficiencies (>93.5% for physically adsorbed Ab and >84.6% for covalently bonded Ab) at 11 cycle aerosol treatment. This CO 2 aerosol treatment did not undermine re-functionalization, which was confirmed by the fluorescent images of FITC–Abs for fresh and reused chips. Desorption of the immobilized layers was validated by Fourier transform infrared and X-ray photoelectron spectroscopic analyses. We also conducted an experiment on the regeneration of E. coli sensing chips using this aerosol treatment, and the chips were re-used 5 times successfully. This mechanical desorption technique is a highly effective and novel strategy for reusable biosensors

  17. Detection of Metallothionein in Javanese Medaka (Oryzias javanicus, Using a scFv-Immobilized Protein Chip

    Directory of Open Access Journals (Sweden)

    Euiyeon Lee

    2018-04-01

    Full Text Available Environmental pollution by various industrial chemicals and biological agents poses serious risks to human health. Especially, marine contamination by potentially toxic elements (PTEs has become a global concern in recent years. Many efforts have been undertaken to monitor the PTE contamination of the aquatic environment. However, there are few approaches available to assess the PTE exposure of aquatic organisms. In this research, we developed a strategy to evaluate the heavy metal exposure of marine organisms, by measuring the expression levels of metallothionein protein derived from Oryzias javanicus (OjaMT. OjaMT is a biomarker of heavy metal exposure because the expression level increases upon heavy metal exposure. The developed assay is based on a real-time, label-free surface plasmon resonance (SPR measurement. Anti-OjaMT antibody and anti-OjaMT single-chain fragment of variable region (scFv were used as detection probes. Two types of SPR sensor chips were fabricated, by immobilizing antibody or Cys3-tagged scFv (scFv-Cys3 in a controlled orientation and were tested for in situ label-free OjaMT detection. Compared to the antibody-presenting sensor chips, the scFv-presenting sensor chips showed improved performance, displaying enhanced sensitivity and enabling semi-quantitative detection. The portable SPR system combined with scFv-immobilized sensor chips is expected to provide an excellent point-of-care testing system that can monitor target biomarkers in real time.

  18. Plasma Surface Modification for Immobilization of Bone Morphogenic Protein-2 on Polycaprolactone Scaffolds

    Science.gov (United States)

    Kim, Byung Hoon; Myung, Sung Woon; Jung, Sang Chul; Ko, Yeong Mu

    2013-11-01

    The immobilization of recombinant human bone formation protein-2 (rhBMP-2) on polycaprolactone (PCL) scaffolds was performed by plasma polymerization. RhBMP-2, which induces osteoblast differentiation in various cell types, is a growth factor that plays an important role in bone formation and repair. The surface of the PCL scaffold was functionalized with the carboxyl groups of plasma-polymerized acrylic acid (PPAA) thin films. Plasma polymerization was carried out at a discharge power of 60 W at an acrylic acid flow rate of 7 sccm for 5 min. The PPAA thin film exhibited moderate hydrophilic properties and possessed a high density of carboxyl groups. Carboxyl groups and rhBMP-2 on the PCL scaffolds surface were identified by attenuated total reflection Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, respectively. The alkaline phosphatase activity assay showed that the rhBMP-2 immobilized PCL scaffold increased the level of MG-63 cell differentiation. Plasma surface modification for the preparation of biomaterials, such as biofunctionalized polymer scaffolds, can be used for the binding of bioactive molecules in tissue engineering.

  19. Radiation synthesis of a water-soluble temperature sensitive polymer, activated copolymer and applications in immobilization of proteins

    International Nuclear Information System (INIS)

    Zhai Maolin; Ha Hongfei; Wu Jilan

    1993-01-01

    In this work the radiation polymerization of N-isopropylacrylamide (NIPAAM) in aqueous solutions has been carried out and a water-soluble, temperature sensitive polymer and copolymer were obtained by using γ-rays from Co-60 source at room temperature. We have gained the optimum dose and dose-rate of radiation synthesis of linear polyNIPAAM through determining conversion yield and viscosity. In order to immobilize protein (BSA) and enzyme (HRP) into this water-soluble polymer, we prepared an activated copolymer, poly(N-isopropylacrylamide-co-N-acryloxysuccinimide). The BSA and HRP has been immobilized onto the activated copolymer. The BSA (HRP)/copolymer conjugates still kept the original thermally sensitive properties of the linear polyNIPAAM. The conjugation yield of BSA to the activated copolymer decreased with increasing dose. Immobilized HRP was stable at 0 o C for a long time and has, at least, 4 days stability at room temperature. Immobilized HRP activity was lowered when the temperature was raised. This phenomenon was reversible and the immobilized HRP regained activity. The optimum pH of the immobilized HRP shifted from ca.5 upward to ca. 7. (author)

  20. Effects of 2 weeks lower limb immobilization and two separate rehabilitation regimens on gastrocnemius muscle protein turnover signaling and normalization genes

    DEFF Research Database (Denmark)

    Nedergaard, Anders; Jespersen, Jakob G; Pingel, Jessica

    2012-01-01

    of resistance training and continued protein/carbohydrate supplementation (Study 2). We obtained muscle biopsies from the medial gastrocnemius prior to immobilization (PRE), post-immobilization (IMMO) and post-rehabilitation (REHAB) and measured protein expression and phosphorylation of Akt, mTOR, S6k, 4E-BP1...

  1. Temperature effects on protein depolymerization and amino acid immobilization rates in soils.

    Science.gov (United States)

    Noll, Lisa; Hu, Yuntao; Zhang, Shasha; Zheng, Qing; Wanek, Wolfgang

    2017-04-01

    Increasing N deposition, land use change, elevated atmospheric CO2 concentrations and global warming have altered soil nitrogen (N) cycling during the last decades. Those changes affected ecosystem services, such as C and N sequestration in soils, which calls for a better understanding of soil N transformation processes. The cleavage of macromolecular organic N by extracellular enzymes maintains an ongoing flow of new bioavailable organic N into biotic systems and is considered to be the bottle neck of terrestrial N cycling in litter and soils. Recent studies showed that protein depolymerization is susceptible to changes in C and N availabilities. Based on general biological observations the temperature sensitivity of soil organic N processes is expected to depend on whether they are rather enzyme limited (i.e. Q10=2) or diffusion limited (i.e. Q10= 1.0 - 1.3). However, temperature sensitivities of protein depolymerization and amino acid immobilization are still unknown. We therefore here report short-term temperature effects on organic N transformation rates in soils differing in physicochemical parameters but not in climate. Soil samples were collected from two geologically distinct sites close to the LFZ Raumberg-Gumpenstein, Styria, Austria, each from three different management types (arable land, grassland, forest). Four replicates of mineral soil were taken from every site and management type. The area provides a unique opportunity to study geological and management controls in soils without confounding effects of climate and elevation. The soils differ in several soil chemical parameters, such as soil pH, base saturation, soil C: N ratio and SOM content as well as in soil physical parameters, such as soil texture, bulk density and water holding capacity. Soils were pre-incubated at 5, 15 and 25˚ C for one day. Protein depolymerization rates and amino acid immobilization rates were assessed by an isotope pool dilution assay with 15N labeled amino acids at

  2. Rapid micro-scale proteolysis of proteins for MALDI-MS peptide mapping using immobilized trypsin

    Science.gov (United States)

    Gobom, Johan; Nordhoff, Eckhard; Ekman, Rolf; Roepstorff, Peter

    1997-12-01

    In this study we present a rapid method for tryptic digestion of proteins using micro-columns with enzyme immobilized on perfusion chromatography media. The performance of the method is exemplified with acyl-CoA-binding protein and reduced carbamidomethylated bovine serum albumin. The method proved to be significantly faster and yielded a better sequence coverage and an improved signal-to-noise ratio for the MALDI-MS peptide maps, compared to in-solution- and on-target digestion. Only a single sample transfer step is required, and therefore sample loss due to adsorption to surfaces is reduced, which is a critical issue when handling low picomole to femtomole amounts of proteins. An example is shown with on-column proteolytic digestion and subsequent elution of the digest into a reversed-phase micro-column. This is useful if the sample contains large amounts of salt or is too diluted for MALDI-MS analysis. Furthermore, by step-wise elution from the reversedphase column, a complex digest can be fractionated, which reduces signal suppression and facilitates data interpretation in the subsequent MS-analysis. The method also proved useful for consecutive digestions with enzymes of different cleavage specificity. This is exemplified with on-column tryptic digestion, followed by reversed-phase step-wise elution, and subsequent on-target V8 protease digestion.

  3. Plasma immersion ion implantation of polyurethane shape memory polymer: Surface properties and protein immobilization

    Science.gov (United States)

    Cheng, Xinying; Kondyurin, Alexey; Bao, Shisan; Bilek, Marcela M. M.; Ye, Lin

    2017-09-01

    Polyurethane-type shape memory polymers (SMPU) are promising biomedical implant materials due to their ability to recover to a predetermined shape from a temporary shape induced by thermal activation close to human body temperature and their advantageous mechanical properties including large recovery strains and low recovery stresses. Plasma Immersion Ion Implantation (PIII) is a surface modification process using energetic ions that generates radicals in polymer surfaces leading to carbonisation and oxidation and the ability to covalently immobilise proteins without the need for wet chemistry. Here we show that PIII treatment of SMPU significantly enhances its bioactivity making SMPU suitable for applications in permanent implantable biomedical devices. Scanning Electron Microscopy (SEM), contact angle measurements, surface energy measurements, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) were used to characterise the PIII modified surface, including its after treatment aging kinetics and its capability to covalently immobilise protein directly from solution. The results show a substantial improvement in wettability and dramatic changes of surface chemical composition dependent on treatment duration, due to the generation of radicals and subsequent oxidation. The SMPU surface, PIII treated for 200s, achieved a saturated level of covalently immobilized protein indicating that a full monolayer coverage was achieved. We conclude that PIII is a promising and efficient surface modification method to enhance the biocompatibility of SMPU for use in medical applications that demand bioactivity for tissue integration and stability in vivo.

  4. Thermostable trypsin conjugates immobilized to biogenic magnetite show a high operational stability and remarkable reusability for protein digestion

    Science.gov (United States)

    Pečová, M.; Šebela, M.; Marková, Z.; Poláková, K.; Čuda, J.; Šafářová, K.; Zbořil, R.

    2013-03-01

    In this work, magnetosomes produced by microorganisms were chosen as a suitable magnetic carrier for covalent immobilization of thermostable trypsin conjugates with an expected applicability for efficient and rapid digestion of proteins at elevated temperatures. First, a biogenic magnetite was isolated from Magnetospirillum gryphiswaldense and its free surface was coated with the natural polysaccharide chitosan containing free amino and hydroxy groups. Prior to covalent immobilization, bovine trypsin was modified by conjugating with α-, β- and γ-cyclodextrin. Modified trypsin was bound to the magnetic carriers via amino groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling reagents. The magnetic biomaterial was characterized by magnetometric analysis and electron microscopy. With regard to their biochemical properties, the immobilized trypsin conjugates showed an increased resistance to elevated temperatures, eliminated autolysis, had an unchanged pH optimum and a significant storage stability and reusability. Considering these parameters, the presented enzymatic system exhibits properties that are superior to those of trypsin forms obtained by other frequently used approaches. The proteolytic performance was demonstrated during in-solution digestion of model proteins (horseradish peroxidase, bovine serum albumin and hen egg white lysozyme) followed by mass spectrometry. It is shown that both magnetic immobilization and chemical modification enhance the characteristics of trypsin making it a promising tool for protein digestion.

  5. Immobilization of halophilic Bacillus sp. EMB9 protease on functionalized silica nanoparticles and application in whey protein hydrolysis.

    Science.gov (United States)

    Sinha, Rajeshwari; Khare, S K

    2015-04-01

    The present work targets the fabrication of an active, stable, reusable enzyme preparation using functionalized silica nanoparticles as an effective enzyme support for crude halophilic Bacillus sp. EMB9 protease. The immobilization efficiency under optimized conditions was 60%. Characterization of the immobilized preparation revealed marked increase in pH and thermal stability. It retained 80% of its original activity at 70 °C while t 1/2 at 50 °C showed a five-fold enhancement over that for the free protease. Kinetic constants K m and V max were indicative of a higher reaction velocity along with decreased affinity for substrate. The preparation could be efficiently reused up to 6 times and successfully hydrolysed whey proteins with high degree of hydrolysis. Immobilization of a crude halophilic protease on a nanobased scaffold makes the process cost effective and simple.

  6. Immobilization of flavan-3-ols onto sensor chips to study their interactions with proteins and pectins by SPR

    International Nuclear Information System (INIS)

    Watrelot, Aude A.; Tran, Dong Tien; Buffeteau, Thierry; Deffieux, Denis

    2016-01-01

    Highlights: • Flavanol-macromolecule interactions were determined using SPR. • Flavanols were chemically modified with a linker bearing a thiol group. • Flavanols were immobilized onto a carboxymethyl dextran surface. • Citrus pectin interacted more with flavanols than apple pectin. • Epicatechin interacted more with BSA than flavanol oligomer. - Abstract: Interactions between plant polyphenols and biomacromolecules such as proteins and pectins have been studied by several methods in solution (e.g. isothermal titration calorimetry, dynamic light scattering, nuclear magnetic resonance and spectrophotometry). Herein, these interactions were investigated in real time by Surface Plasmon Resonance (SPR) analysis after immobilization of flavan-3-ols onto a sensor chip surface. (−)-epicatechin, (+)-catechin and flavan-3-ol oligomers with an average degree of polymerization of 2 and 8 were chemically modified using N-(2-(tritylthio)ethyl)propiolamide in order to introduce a spacer unit onto the catecholic B ring. Modified flavan-3-ols were then immobilized onto a carboxymethylated dextran surface (CM5). Immobilization was validated and further verified by evaluating flavan-3-ol interaction with bovine serum albumin (BSA), poly-L-proline or commercial pectins. BSA was found to have a stronger association with monomeric flavan-3-ols than oligomers. SPR analysis of selected flavan-3-ols immobilized onto CM5 sensor chips showed a stronger association for citrus pectins than apple pectins, regardless of flavan-3-ol degree of polymerization.

  7. Immobilization of flavan-3-ols onto sensor chips to study their interactions with proteins and pectins by SPR

    Energy Technology Data Exchange (ETDEWEB)

    Watrelot, Aude A., E-mail: aude.watrelot@avignon.inra.fr [INRA, UMR408 Sécurité et Qualité des Produits d’Origine Végétale, Domaine St Paul, Site Agroparc, 84914 Avignon (France); Université d’Avignon, UMR408 Sécurité et Qualité des Produits d' Origine Végétale, F-84000 Avignon (France); Tran, Dong Tien [Université de Bordeaux, Institut des Sciences Moléculaires (UMR-CNRS 5255), 351 cours de la Libération, 33405 Talence (France); Institut Européen de Chimie et Biologie (IECB), 2 rue Robert Escarpit, 33607 Pessac (France); Buffeteau, Thierry [Université de Bordeaux, Institut des Sciences Moléculaires (UMR-CNRS 5255), 351 cours de la Libération, 33405 Talence (France); Deffieux, Denis [Université de Bordeaux, Institut des Sciences Moléculaires (UMR-CNRS 5255), 351 cours de la Libération, 33405 Talence (France); Institut Européen de Chimie et Biologie (IECB), 2 rue Robert Escarpit, 33607 Pessac (France); and others

    2016-05-15

    Highlights: • Flavanol-macromolecule interactions were determined using SPR. • Flavanols were chemically modified with a linker bearing a thiol group. • Flavanols were immobilized onto a carboxymethyl dextran surface. • Citrus pectin interacted more with flavanols than apple pectin. • Epicatechin interacted more with BSA than flavanol oligomer. - Abstract: Interactions between plant polyphenols and biomacromolecules such as proteins and pectins have been studied by several methods in solution (e.g. isothermal titration calorimetry, dynamic light scattering, nuclear magnetic resonance and spectrophotometry). Herein, these interactions were investigated in real time by Surface Plasmon Resonance (SPR) analysis after immobilization of flavan-3-ols onto a sensor chip surface. (−)-epicatechin, (+)-catechin and flavan-3-ol oligomers with an average degree of polymerization of 2 and 8 were chemically modified using N-(2-(tritylthio)ethyl)propiolamide in order to introduce a spacer unit onto the catecholic B ring. Modified flavan-3-ols were then immobilized onto a carboxymethylated dextran surface (CM5). Immobilization was validated and further verified by evaluating flavan-3-ol interaction with bovine serum albumin (BSA), poly-L-proline or commercial pectins. BSA was found to have a stronger association with monomeric flavan-3-ols than oligomers. SPR analysis of selected flavan-3-ols immobilized onto CM5 sensor chips showed a stronger association for citrus pectins than apple pectins, regardless of flavan-3-ol degree of polymerization.

  8. Effect of plaster cast immobilization on the turnover rates of soluble proteins and lactate dehydrogenase isoenzymes of rabbit M. soleus

    Energy Technology Data Exchange (ETDEWEB)

    Edes, I.; Dosa, E.; Sohar, I.; Guba, F. (Orvostudomanyi Egyetem, Szeged (Hungary). Biokemiai Tanszek)

    1982-01-01

    In atrophized muscle the decreases of the activity of LDH isoenzymes can be explained partly by a 15 per cent decrease of the enzyme synthesis and partly by a 25 per cent increase in catabolism. The quantities of the soluble proteins and LDH were measured after intravenously administered /sup 3/H-leucin incorporation, from the musculus soleus. LDH was isolated by means of affinity chromatography. Radioactivity was determined in a Packard Tri-Carb scintillation counter. The synthesis rate of soluble proteins barely changed during immobilization. In the atrophized muscle the decrease of the amount of soluble proteins could be almost exclusively interpreted in terms of a 25 per cent enchancement of degradative process. The accelerated catabolism is most probably due to the proteolytic enzymes activated by immobilization.

  9. Applications of lipid based formulation technologies in the delivery of biotechnology-based therapeutics.

    Science.gov (United States)

    du Plessis, Lissinda H; Marais, Etienne B; Mohammed, Faruq; Kotzé, Awie F

    2014-01-01

    In the last decades several new biotechnologically-based therapeutics have been developed due to progress in genetic engineering. A growing challenge facing pharmaceutical scientists is formulating these compounds into oral dosage forms with adequate bioavailability. An increasingly popular approach to formulate biotechnology-based therapeutics is the use of lipid based formulation technologies. This review highlights the importance of lipid based drug delivery systems in the formulation of oral biotechnology based therapeutics including peptides, proteins, DNA, siRNA and vaccines. The different production procedures used to achieve high encapsulation efficiencies of the bioactives are discussed, as well as the factors influencing the choice of excipient. Lipid based colloidal drug delivery systems including liposomes and solid lipid nanoparticles are reviewed with a focus on recent advances and updates. We further describe microemulsions and self-emulsifying drug delivery systems and recent findings on bioactive delivery. We conclude the review with a few examples on novel lipid based formulation technologies.

  10. ANALYSIS OF DRUG-PROTEIN BINDING BY ULTRAFAST AFFINITY CHROMATOGRAPHY USING IMMOBILIZED HUMAN SERUM ALBUMIN

    Science.gov (United States)

    Mallik, Rangan; Yoo, Michelle J.; Briscoe, Chad J.; Hage, David S.

    2010-01-01

    Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug binding studies and in the high-throughput screening of new drug candidates. PMID:20227701

  11. Modulation of Protein Adsorption and Cell Proliferation on Polyethylene Immobilized Graphene Oxide Reinforced HDPE Bionanocomposites.

    Science.gov (United States)

    Upadhyay, Rahul; Naskar, Sharmistha; Bhaskar, Nitu; Bose, Suryasarathi; Basu, Bikramjit

    2016-05-18

    The uniform dispersion of nanoparticles in a polymer matrix, together with an enhancement of interfacial adhesion is indispensable toward achieving better mechanical properties in the nanocomposites. In the context to biomedical applications, the type and amount of nanoparticles can potentially influence the biocompatibility. To address these issues, we prepared high-density polyethylene (HDPE) based composites reinforced with graphene oxide (GO) by melt mixing followed by compression molding. In an attempt to tailor the dispersion and to improve the interfacial adhesion, we immobilized polyethylene (PE) onto GO sheets by nucleophilic addition-elimination reaction. A good combination of yield strength (ca. 20 MPa), elastic modulus (ca. 600 MPa), and an outstanding elongation at failure (ca. 70%) were recorded with 3 wt % polyethylene grafted graphene oxide (PE-g-GO) reinforced HDPE composites. Considering the relevance of protein adsorption as a biophysical precursor to cell adhesion, the protein adsorption isotherms of bovine serum albumin (BSA) were determined to realize three times higher equilibrium constant (Keq) for PE-g-GO-reinforced HDPE composites as compared to GO-reinforced composites. To assess the cytocompatibility, we grew osteoblast cell line (MC3T3) and human mesenchymal stem cells (hMSCs) on HDPE/GO and HDPE/PE-g-GO composites, in vitro. The statistically significant increase in metabolically active cell over different time periods in culture for up to 6 days in MC3T3 and 7 days for hMSCs was observed, irrespective of the substrate composition. Such observation indicated that HDPE with GO or PE-g-GO addition (up to 3 wt %) can be used as cell growth substrate. The extensive proliferation of cells with oriented growth pattern also supported the fact that tailored GO addition can support cellular functionality in vitro. Taken together, the experimental results suggest that the PE-g-GO in HDPE can effectively be utilized to enhance both mechanical and

  12. Experimental Study of the Oriented Immobilization of Antibodies on Photonic Sensing Structures by Using Protein A as an Intermediate Layer

    Directory of Open Access Journals (Sweden)

    Raffaele Caroselli

    2018-03-01

    Full Text Available A proper antibody immobilization on a biosensor is a crucial step in order to obtain a high sensitivity to be able to detect low target analyte concentrations. In this paper, we present an experimental study of the immobilization process of antibodies as bioreceptors on a photonic ring resonator sensor. A protein A intermediate layer was created on the sensor surface in order to obtain an oriented immobilization of the antibodies, which enhances the interaction with the target antigens to be detected. The anti-bovine serum albumin (antiBSA-bovine serum albumin (BSA pair was used as a model for our study. An opto-fluidic setup was developed in order to flow the different reagents and, simultaneously, to monitor in real-time the spectral response of the photonic sensing structure. The antiBSA immobilization and the BSA detection, their repeatability, and specificity were studied in different conditions of the sensor surface. Finally, an experimental limit of detection for BSA recognition of only 1 ng/mL was obtained.

  13. In vitro and in vivo evaluation of bone morphogenetic protein-2 (BMP-2) immobilized collagen-coated polyetheretherketone (PEEK)

    Science.gov (United States)

    Du, Ya-Wei; Zhang, Li-Nan; Ye, Xin; Nie, He-Min; Hou, Zeng-Tao; Zeng, Teng-Hui; Yan, Guo-Ping; Shang, Peng

    2015-03-01

    Polyetheretherketone (PEEK) is regarded as one of the most potential candidates of biomaterials in spinal implant applications. However, as a bioinert material, PEEK plays a limited role in osteoconduction and osseointegration. In this study, recombinant human bone morphogenetic protein-2 (rhBMP-2) was immobilized onto the surface of collagen-coated PEEK in order to prepare a multi-functional material. After adsorbed onto the PEEK surface by hydrophobic interaction, collagen was cross-linked with N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). EDC/NHS system also contributed to the immobilization of rhBMP-2. Water contact angle tests, XPS and SEM clearly demonstrated the surface changes. ELISA tests quantified the amount of rhBMP-2 immobilized and the release over a period of 30 d. In vitro evaluation proved that the osteogenesis differentiation rate was higher when cells were cultured on modified PEEK discs than on regular ones. In vivo tests were conducted and positive changes of major parameters were presented. This report demonstrates that the rhBMP-2 immobilized method for PEEK modification increase bioactivity in vitro and in vivo, suggesting its practicability in orthopedic and spinal clinical applications.

  14. Phosphopeptide Enrichment by Covalent Chromatography after Derivatization of Protein Digests Immobilized on Reversed-Phase Supports

    Science.gov (United States)

    Nika, Heinz; Nieves, Edward; Hawke, David H.; Angeletti, Ruth Hogue

    2013-01-01

    A rugged sample-preparation method for comprehensive affinity enrichment of phosphopeptides from protein digests has been developed. The method uses a series of chemical reactions to incorporate efficiently and specifically a thiol-functionalized affinity tag into the analyte by barium hydroxide catalyzed β-elimination with Michael addition using 2-aminoethanethiol as nucleophile and subsequent thiolation of the resulting amino group with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. Gentle oxidation of cysteine residues, followed by acetylation of α- and ε-amino groups before these reactions, ensured selectivity of reversible capture of the modified phosphopeptides by covalent chromatography on activated thiol sepharose. The use of C18 reversed-phase supports as a miniaturized reaction bed facilitated optimization of the individual modification steps for throughput and completeness of derivatization. Reagents were exchanged directly on the supports, eliminating sample transfer between the reaction steps and thus, allowing the immobilized analyte to be carried through the multistep reaction scheme with minimal sample loss. The use of this sample-preparation method for phosphopeptide enrichment was demonstrated with low-level amounts of in-gel-digested protein. As applied to tryptic digests of α-S1- and β-casein, the method enabled the enrichment and detection of the phosphorylated peptides contained in the mixture, including the tetraphosphorylated species of β-casein, which has escaped chemical procedures reported previously. The isolates proved highly suitable for mapping the sites of phosphorylation by collisionally induced dissociation. β-Elimination, with consecutive Michael addition, expanded the use of the solid-phase-based enrichment strategy to phosphothreonyl peptides and to phosphoseryl/phosphothreonyl peptides derived from proline-directed kinase substrates and to their O-sulfono- and O-linked β-N-acetylglucosamine (O

  15. Efficient protein immobilization on polyethersolfone electrospun nanofibrous membrane via covalent binding for biosensing applications

    Energy Technology Data Exchange (ETDEWEB)

    Mahmoudifard, Matin [Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran (Iran, Islamic Republic of); Soudi, Sara [Stem Cell Biology Department, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Soleimani, Masoud [Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Hosseinzadeh, Simzar [Nanotechnology and Tissue Engineering Department, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Esmaeili, Elaheh [Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran (Iran, Islamic Republic of); Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Vossoughi, Manouchehr, E-mail: vosoughi@sharif.edu [Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran (Iran, Islamic Republic of); Chemical and Petroleum Engineering Department, Sharif University of Technology, Tehran (Iran, Islamic Republic of)

    2016-01-01

    In this paper we introduce novel strategy for antibody immobilization using high surface area electrospun nanofibrous membrane based on ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling chemistry. To present the high performance of proposed biosensors, anti-staphylococcus enterotoxin B (anti-SEB) was used as a model to demonstrate the utility of our proposed system. Polymer solution of polyethersolfone was used to fabricate fine nanofibrous membrane. Moreover, industrial polyvinylidene fluoride membrane and conventional microtiter plate were also used to compare the efficiency of antibody immobilization. Scanning electron microscopy images were taken to study the morphology of the membranes. The surface activation of nanofibrous membrane was done with the help of O{sub 2} plasma. PES nanofibrous membrane with carboxyl functional groups for covalent attachment of antibodies were treated by EDC/NHS coupling agent. The quantity of antibody immobilization was measured by enzyme-linked immuno sorbent assay (ELISA) method. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy was performed to confirm the covalent immobilization of antibody on membrane. Atomic force microscopy, scanning electron microscopy and invert fluorescence microscopy were used to analyze the antibody distribution pattern on solid surfaces. Results show that oxygen plasma treatment effectively increased the amount of antibody immobilization through EDC/NHS coupling chemistry. It was found that the use of nanofibrous membrane causes the improved detection signal of ELISA based biosensors in comparison to the standard assay carried out in the 96-well microtiter plate. This method has the potential to improve the ELISA-based biosensor and we believe that this technique can be used in various biosensing methods. - Highlights: • Introduction of novel strategy for antibody immobilization using high surface area electrospun

  16. Pharmaceutical and biomedical applications of lipid-based nanocarriers.

    Science.gov (United States)

    Carbone, Claudia; Leonardi, Antonio; Cupri, Sarha; Puglisi, Giovanni; Pignatello, Rosario

    2014-03-01

    Increasing attention is being given to lipid nanocarriers (LNs) as drug delivery systems, due to the advantages offered of a higher biocompatibility and lower toxicity compared with polymeric nanoparticles. Many administration routes are being investigated for LNs, including topical, oral and parenteral ones. LNs are also proposed for specific applications such as cancer treatment, gene therapy, diagnosis and medical devices production. However, the high number of published research articles does not match an equal amount of patents. A recent Review of ours, published in Pharmaceutical Patent Analyst, reported the patents proposing novel methods for the production of LNs. This review work discusses recent patents, filed in 2007-2013 and dealing with the industrial applications of lipid-based nanocarriers for the vectorization of therapeutically relevant molecules, as well as biotech products such as proteins, gene material and vaccines, in the pharmaceutical, diagnostic and biomedical areas.

  17. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    Science.gov (United States)

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.

  18. Immobilized Metal Affinity Chromatography Co-Purifies TGF-β1 with Histidine-Tagged Recombinant Extracellular Proteins

    Science.gov (United States)

    Kaur, Jasvir; Reinhardt, Dieter P.

    2012-01-01

    Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC). Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls. PMID:23119075

  19. Dependence of protein binding capacity of dimethylamino-γ-butyric-acid (DMGABA)-immobilized porous membrane on composition of solvent used for DMGABA immobilization

    International Nuclear Information System (INIS)

    Iwanade, Akio; Umeno, Daisuke; Saito, Kyoichi; Sugo, Takanobu

    2013-01-01

    Dimethylamino-γ-butyric acid (DMGABA) as an ampholite was reacted with the epoxy group of the poly-glycidyl methacrylate chain grafted onto the pore surface of a porous hollow-fiber polyethylene membrane by radiation-induced graft polymerization. DMGABA was dissolved in a mixture of dioxane and water at various dioxane volume fractions, defined by dividing the dioxane volume by the total volume. The equilibrium binding capacity (EBC) of the DMGABA-immobilized porous hollow-fiber membrane for lysozyme was evaluated in the permeation mode. The EBC was varied from a 1/50-fold monolayer binding capacity to a 10-fold monolayer binding capacity by controlling the composition of the solvent used for DMGABA immobilization and the molar conversion of the epoxy group into the DMGABA group. - Highlights: ► A DMGABA membrane was immobilized by irradiation induced graft polymerization. ► The DMGABA was immobilized in a mixture of dioxane and water of various compositions. ► Lysozyme adsorptivity of DMGABA-immobilized membranes evaluated in the permeation mode. ► The composition of the DMGABA immobilized solvent can control adsorptivity

  20. Myristoylation as a general method for immobilization and alignment of soluble proteins for solid-state NMR structural studies

    International Nuclear Information System (INIS)

    Mesleh, M.F.; Valentine, K.G.; Opella, S.J.; Louis, J.M.; Gronenborn, A.M.

    2003-01-01

    N-terminal myristoylation of the immunoglobulin-binding domain of protein G (GB1) from group G Streptococcus provides the means to bind the protein to aligned phospholipid bilayers for solid-state NMR structural studies. The myristoylated protein is immobilized by its interactions with bilayers, and the sample alignment enables orientationally dependent 15 N chemical shifts and 1 H- 15 N-dipolar couplings to be measured. Spectra calculated for the average solution NMR structure of the protein at various orientations with respect to the magnetic field direction were compared to the experimental spectrum. The best fit identified the orientation of the myristoylated protein on the lipid bilayers, and demonstrated that the protein adopts a similar structure in both its myristoylated and non-myristoylated forms, and that the structure is not grossly distorted by its interaction with the phosholipid bilayer surface or by its location in the restricted aqueous space between bilayer leaflets. The protein is oriented such that its charged sides face the phosphatidylcholine headgroups of the lipids with the single amphiphilic helix running parallel to the bilayer surface

  1. Surface Immobilization of Human Arginase-1 with an Engineered Ice Nucleation Protein Display System in E. coli.

    Directory of Open Access Journals (Sweden)

    Zhen Zhang

    Full Text Available Ice nucleation protein (INP is frequently used as a surface anchor for protein display in gram-negative bacteria. Here, MalE and TorA signal peptides, and three charged polypeptides, 6×Lys, 6×Glu and 6×Asp, were anchored to the N-terminus of truncated INP (InaK-N to improve its surface display efficiency for human Arginase1 (ARG1. Our results indicated that the TorA signal peptide increased the surface translocation of non-protein fused InaK-N and human ARG1 fused InaK-N (InaK-N/ARG1 by 80.7% and 122.4%, respectively. Comparably, the MalE signal peptide decreased the display efficiencies of both the non-protein fused InaK-N and InaK-N/ARG1. Our results also suggested that the 6×Lys polypeptide significantly increased the surface display efficiency of K6-InaK-N/ARG1 by almost 2-fold, while also practically abolishing the surface translocation of non-protein fused InaK-N, indicating the interesting roles of charged polypeptides in bacteria surface display systems. Cell surface-immobilized K6-InaK-N/ARG1 presented an arginase activity of 10.7 U/OD600 under the optimized conditions of 40°C, pH 10.0 and 1 mM Mn2+, which could convert more than 95% of L-Arginine (L-Arg to L-Ornithine (L-Orn in 16 hours. The engineered InaK-Ns expanded the INP surface display system, which aided in the surface immobilization of human ARG1 in E. coli cells.

  2. Immobilization of immunoglobulin G in a highly oriented manner on a protein-A terminated multilayer system

    Energy Technology Data Exchange (ETDEWEB)

    Zengin, Adem [Department of Chemistry, Faculty of Art and Science, Gazi University, 06500 Besevler, Ankara (Turkey); Caykara, Tuncer, E-mail: caykara@gazi.edu.tr [Department of Chemistry, Faculty of Art and Science, Gazi University, 06500 Besevler, Ankara (Turkey)

    2011-01-01

    In this study, we have fabricated a multilayer system consisting of 3-glycidoxypropyldimethylmethoxysilane (GPDS), poly(dimethylsiloxane) bis 3-aminopropyl terminated (PDMS) and protein-A on a silicon wafer surface for oriented immobilization of immunoglobilin G (IgG). The multilayer system with a different component in each layer was characterized by ellipsometry, contact-angle goniometer, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) and fluorescence microscopy. The epoxy-terminated monolayer was formed by the chemisorption of GPDS molecules on the hydroxylated silicon surface. The PDMS film about 4.5 nm thick was produced on the GPDS-monolayer by the chemical reaction between the amine groups at the end of PDMS chain and the epoxy groups of GPDS molecules. By introducing the PDMS chains, the hydrophilic character of GPDS-monolayer decreased. Study of the time dependence of polymer grafting showed that the chemisorption of GPDS is fast, whereas at least 16 h is needed to generate the homogeneous PDMS layer. For immobilization of IgG molecules in a highly oriented manner, protein-A molecules were first chemically bound to an ultrathin ({approx}4.5 nm) PDMS reactive polymer layer and later used to capture IgG. It was shown that the existence of protein-A in the multilayer system has a strong influence on the binding properties of IgG not only in the efficiency of binding, but also in its specificity. In conclusion, the multilayer system with protein-A has the potential to be further developed into an efficient immunoassay protein chip.

  3. Correlation between cationic lipid-based transfection and cell division

    Energy Technology Data Exchange (ETDEWEB)

    Kirchenbuechler, Inka; Kirchenbuechler, David; Elbaum, Michael, E-mail: michael@elbaum.ac.il

    2016-07-01

    We evaluate the temporal relation between protein expression by cationic lipid-mediated transfection and cell division using time lapse fluorescence microscopy. Detailed image analysis provides new insights on the single cell level while simultaneously achieving appropriate statistics. Earlier evidence by less direct methods such as flow cytometry indicates a primary route for transfection involving nuclear envelope breakdown, but also suggests the existence of a pathway independent of mitosis. We confirm and quantify both mechanisms. We found the timing for successful transfection to be unexpectedly flexible, contrary to assertions of a narrow time window. Specifically, cells dividing more than 24 h after exposure to the transfection medium express the probed protein at a comparable level to cells in a mitotic state during or shortly after transfection. This finding can have a profound impact on the guidance and development of non-viral gene delivery materials. - Highlights: • Cationic lipid-based transfection supports protein expression without cell division. • Protein expression is unrelated to cell cycle status at the time of transfection. • Time-lapse imaging provides direct evaluation without statistical averaging. • Lipoplex dissociation is a likely target for improvement of transfection efficiency.

  4. Peptide aptamer-assisted immobilization of green fluorescent protein for creating biomolecule-complexed carbon nanotube device

    Science.gov (United States)

    Nii, Daisuke; Nozawa, Yosuke; Miyachi, Mariko; Yamanoi, Yoshinori; Nishihara, Hiroshi; Tomo, Tatsuya; Shimada, Yuichiro

    2017-10-01

    Carbon nanotubes are a novel material for next-generation applications. In this study, we generated carbon nanotube and green fluorescent protein (GFP) conjugates using affinity binding peptides. The carbon nanotube-binding motif was introduced into the N-terminus of the GFP through molecular biology methods. Multiple GFPs were successfully aligned on a single-walled carbon nanotube via the molecular recognition function of the peptide aptamer, which was confirmed through transmission electron microscopy and optical analysis. Fluorescence spectral analysis results also suggested that the carbon nanotube-GFP complex was autonomously formed with orientation and without causing protein denaturation during immobilization. This simple process has a widespread potential for fabricating carbon nanotube-biomolecule hybrid devices.

  5. Pepsin immobilized in dextran-modified fused-silica capillaries for on-line protein digestion and peptide mapping.

    Science.gov (United States)

    Stigter, E C A; de Jong, G J; van Bennekom, W P

    2008-07-07

    On-line digestion of proteins under acidic conditions was studied using micro-reactors consisting of dextran-modified fused-silica capillaries with covalently immobilized pepsin. The proteins used in this study differed in molecular weight, isoelectric point and sample composition. The injected protein samples were completely digested in 3 min and the digest was analyzed with micro-high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). The different proteins present in the samples could be identified with a Mascot database search on the basis of auto-MS/MS data. It proved also to be possible to digest and analyze protein mixtures with a sequence coverage of 55% and 97% for the haemoglobin beta- and alpha-chain, respectively, and 35-55% for the various casein variants. Protease auto-digestion, sample carry-over and loss of signal due to adsorption of the injected proteins were not observed. The backpressure of the reactor is low which makes coupling to systems such as Surface Plasmon Resonance biosensors, which do not tolerate too high pressure, possible. The reactor was stable for at least 40 days when used continuously.

  6. Pepsin immobilized in dextran-modified fused-silica capillaries for on-line protein digestion and peptide mapping

    Energy Technology Data Exchange (ETDEWEB)

    Stigter, E.C.A. [Division of Biomedical Analysis, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht (Netherlands)], E-mail: e.c.a.stigter@uu.nl; Jong, G.J. de; Bennekom, W.P. van [Division of Biomedical Analysis, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht (Netherlands)

    2008-07-07

    On-line digestion of proteins under acidic conditions was studied using micro-reactors consisting of dextran-modified fused-silica capillaries with covalently immobilized pepsin. The proteins used in this study differed in molecular weight, isoelectric point and sample composition. The injected protein samples were completely digested in 3 min and the digest was analyzed with micro-high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). The different proteins present in the samples could be identified with a Mascot database search on the basis of auto-MS/MS data. It proved also to be possible to digest and analyze protein mixtures with a sequence coverage of 55% and 97% for the haemoglobin {beta}- and {alpha}-chain, respectively, and 35-55% for the various casein variants. Protease auto-digestion, sample carry-over and loss of signal due to adsorption of the injected proteins were not observed. The backpressure of the reactor is low which makes coupling to systems such as Surface Plasmon Resonance biosensors, which do not tolerate too high pressure, possible. The reactor was stable for at least 40 days when used continuously.

  7. Bioactivity of immobilized hyaluronic acid derivatives regarding protein adsorption and cell adhesion

    DEFF Research Database (Denmark)

    Köwitsch, Alexander; Yang, Yuan; Ma, Ning

    2011-01-01

    with HA on physicochemical surface properties of these substrata and estimates of the quantities of immobilized HA were obtained by different physical methods such as contact angle measurements, ellipsometry, and atomic force microscopy. The bioactivity of aHA and tHA toward their natural binding partner...... affects cell growth and differentiation. A lower number and spreading of cells were observed on HA-modified surfaces compared to amino- and vinyl-terminated glass and silicon surfaces. Immunofluorescence microscopy also revealed that adhesion of fibroblast plated on HA-modified surfaces was mediated...... primarily by HA receptor CD44, indicating that bioactivity of HA was not significantly reduced by chemical modification....

  8. Direct Detection of Protein Biomarkers in Human Fluids Using Site-Specific Antibody Immobilization Strategies

    Directory of Open Access Journals (Sweden)

    Maria Soler

    2014-01-01

    Full Text Available Design of an optimal surface biofunctionalization still remains an important challenge for the application of biosensors in clinical practice and therapeutic follow-up. Optical biosensors offer real-time monitoring and highly sensitive label-free analysis, along with great potential to be transferred to portable devices. When applied in direct immunoassays, their analytical features depend strongly on the antibody immobilization strategy. A strategy for correct immobilization of antibodies based on the use of ProLinker™ has been evaluated and optimized in terms of sensitivity, selectivity, stability and reproducibility. Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities. ProLinker™-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum. Furthermore, we have implemented the ProLinker™ strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis.

  9. Direct detection of protein biomarkers in human fluids using site-specific antibody immobilization strategies.

    Science.gov (United States)

    Soler, Maria; Estevez, M-Carmen; Alvarez, Mar; Otte, Marinus A; Sepulveda, Borja; Lechuga, Laura M

    2014-01-29

    Design of an optimal surface biofunctionalization still remains an important challenge for the application of biosensors in clinical practice and therapeutic follow-up. Optical biosensors offer real-time monitoring and highly sensitive label-free analysis, along with great potential to be transferred to portable devices. When applied in direct immunoassays, their analytical features depend strongly on the antibody immobilization strategy. A strategy for correct immobilization of antibodies based on the use of ProLinker™ has been evaluated and optimized in terms of sensitivity, selectivity, stability and reproducibility. Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities. ProLinker™-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum. Furthermore, we have implemented the ProLinker™ strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis.

  10. Engineering cholesterol-based fibers for antibody immobilization and cell capture

    Science.gov (United States)

    Cohn, Celine

    In 2015, the United States is expected to have nearly 600,000 deaths attributed to cancer. Of these 600,000 deaths, 90% will be a direct result of cancer metastasis, the spread of cancer throughout the body. During cancer metastasis, circulating tumor cells (CTCs) are shed from primary tumors and migrate through bodily fluids, establishing secondary cancer sites. As cancer metastasis is incredibly lethal, there is a growing emphasis on developing "liquid biopsies" that can screen peripheral blood, search for and identify CTCs. One popular method for capturing CTCs is the use of a detection platform with antibodies specifically suited to recognize and capture cancer cells. These antibodies are immobilized onto the platform and can then bind and capture cells of interest. However, current means to immobilize antibodies often leave them with drastically reduced function. The antibodies are left poorly suited for cell capture, resulting in low cell capture efficiencies. This body of work investigates the use of lipid-based fibers to immobilize proteins in a way that retains protein function, ultimately leading to increased cell capture efficiencies. The resulting increased efficiencies are thought to arise from the retained three-dimensional structure of the protein as well as having a complete coating of the material surface with antibodies that are capable of interacting with their antigens. It is possible to electrospin cholesterol-based fibers that are similar in design to the natural cell membrane, providing proteins a more natural setting during immobilization. Such fibers have been produced from cholesterol-based cholesteryl succinyl silane (CSS). These fibers have previously illustrated a keen aptitude for retaining protein function and increasing cell capture. Herein the work focuses on three key concepts. First, a model is developed to understand the immobilization mechanism used by electrospun CSS fibers. The antibody immobilization and cell capturing

  11. Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Shaw, A.; Larsen, M.; Roepstorff, P.

    1999-01-01

    magnitude of IFN-gamma responsive genes has been reported previously. Our goal is to identify and map IFN-gamma-regulated HeLa cell proteins to the two-dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system...

  12. Actin Immobilization on Chitin for Purifying Myosin II: A Laboratory Exercise That Integrates Concepts of Molecular Cell Biology and Protein Chemistry

    Science.gov (United States)

    de Souza, Marcelle Gomes; Grossi, Andre Luiz; Pereira, Elisangela Lima Bastos; da Cruz, Carolina Oliveira; Mendes, Fernanda Machado; Cameron, Luiz Claudio; Paiva, Carmen Lucia Antao

    2008-01-01

    This article presents our experience on teaching biochemical sciences through an innovative approach that integrates concepts of molecular cell biology and protein chemistry. This original laboratory exercise is based on the preparation of an affinity chromatography column containing F-actin molecules immobilized on chitin particles for purifying…

  13. Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

    Science.gov (United States)

    Postupalenko, Viktoriia; Desplancq, Dominique; Orlov, Igor; Arntz, Youri; Spehner, Danièle; Mely, Yves; Klaholz, Bruno P; Schultz, Patrick; Weiss, Etienne; Zuber, Guy

    2015-09-01

    Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. AN INTEGRATIVE WAY OF TEACHING MOLECULAR CELL BIOLOGY AND PROTEIN CHEMISTRY USING ACTIN IMMOBILIZATION ON CHITIN FOR PURIFYING MYOSIN II.

    Directory of Open Access Journals (Sweden)

    M.G. Souza

    2007-05-01

    Full Text Available Our intent is to present our experience on teaching Molecular Cell Biology andProtein Chemistry at UNIRIO through an innovative approach that includes myosin IIextraction and purification. We took advantage of the properties of muscle contractionand propose a simple method for purifying myosin II by affinity chromatography. Thisoriginal method is based on the preparation of an affinity column containing actinmolecules covalently bound to chitin particles. We propose a three-week syllabus thatincludes lectures and bench experimental work. The syllabus favors the activelearning of protein extraction and purification, as well as, of scientific concepts suchas muscle contraction, cytoskeleton structure and its importance for the living cell. Italso promotes the learning of the biotechnological applications of chitin and theapplications of protein immobilization in different industrial fields. Furthermore, theactivities also target the development of laboratorial technical abilities, thedevelopment of problem solving skills and the ability to write up a scientific reportfollowing the model of a scientific article. It is very important to mention that thissyllabus can be used even in places where a facility such as ultra-centrifugation islacking.

  15. Nucleoside-Lipid-Based Nanocarriers for Sorafenib Delivery

    Science.gov (United States)

    Benizri, Sebastien; Ferey, Ludivine; Alies, Bruno; Mebarek, Naila; Vacher, Gaelle; Appavoo, Ananda; Staedel, Cathy; Gaudin, Karen; Barthélémy, Philippe

    2018-01-01

    Although the application of sorafenib, a small inhibitor of tyrosine protein kinases, to cancer treatments remains a worldwide option in chemotherapy, novel strategies are needed to address the low water solubility (drug. In this context, the use of nanocarriers is currently investigated in order to overcome these drawbacks. In this contribution, we report a new type of sorafenib-based nanoparticles stabilized by hybrid nucleoside-lipids. The solid lipid nanoparticles (SLNs) showed negative or positive zeta potential values depending on the nucleoside-lipid charge. Transmission electron microscopy of sorafenib-loaded SLNs revealed parallelepiped nanoparticles of about 200 nm. Biological studies achieved on four different cell lines, including liver and breast cancers, revealed enhanced anticancer activities of Sorafenib-based SLNs compared to the free drug. Importantly, contrast phase microscopy images recorded after incubation of cancer cells in the presence of SLNs at high concentration in sorafenib (> 80 μM) revealed a total cancer cell death in all cases. These results highlight the potential of nucleoside-lipid-based SLNs as drug delivery systems.

  16. Electrochemical and spectroscopic investigations of immobilized de novo designed heme proteins on metal electrodes

    DEFF Research Database (Denmark)

    Albrecht, Tim; Li, WW; Ulstrup, Jens

    2005-01-01

    On the basis of rational design principles, template-assisted four-helix-bundle proteins that include two histidines for coordinative binding of a heme were synthesized. Spectroscopic and thermodynamic characterization of the proteins in solution reveals the expected bis-histidine coordinated heme...

  17. Surface functionalization of superparamagnetic nanoparticles encapsulated by chitosan for protein immobilization; Funcionalizacao da superficie de nanoparticulas superparamagneticas encapsuladas por quitosana para a imobilizacao de proteinas

    Energy Technology Data Exchange (ETDEWEB)

    Sousa, Jose Silva de

    2010-07-01

    Nanoscience and nanotechnology have opened up numerous developments of devices and systems on the nanometer scale, with new molecular organization, properties and functions. In this context, the polymeric magnetic nanoparticles are composites formed by magnetic materials with a particle size between 1 and 100 nm combined with functional polymers. They are well-known and have been widely studied because of its applications in various technology areas. Applications on the biological and medical areas include separation and immobilization of enzymes and proteins, improved techniques of magnetic resonance imaging and diagnostic systems for controlled drug delivery. In this work, proteins were immobilized on the surface of a biopolymer combined with superparamagnetic particles of magnetite. The biopolymer chitosan was used, cross-linked and functionalized with glutaraldehyde, applicable to the biological assays. Three types of magnetic composites were obtained, which were called QM1Glu, QM2NaGlu and QM3Glu. They were characterized by X-ray diffraction, scanning electron microscopy, vibrating sample magnetometry, differential scanning calorimetry, thermogravimetry and infrared spectroscopy. They were evaluated concerning the immobilization of the proteins bovine serum albumin (BSA), collagen and trypsin. The study showed that the immobilization of proteins on the biopolymer occurred in 30 min of incubation. The magnetic composite of non functionalized chitosan (QM3) was also evaluated. For trypsin, it was found that the immobilization potential of QM3 was higher than that observed for QM3Glu. After 30 days, the trypsin of the QM3-Trip and QM3Glu-Trip was still with activity. The activity and the enzyme kinetics of the QM3Glu-Trip with the substrate BApNA were demonstrated. (author)

  18. Correlation of acidic and basic carrier ampholyte and immobilized pH gradient two-dimensional gel electrophoresis patterns based on mass spectrometric protein identification

    DEFF Research Database (Denmark)

    Nawrocki, A; Larsen, Martin Røssel; Podtelejnikov, A V

    1998-01-01

    Separation of proteins on either carrier ampholyte-based or immobilized pH gradient-based two-dimensional (2-D) gels gives rise to electrophoretic patterns that are difficult to compare visually. In this paper we have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI......-MS) to determine the identities of 335 protein spots in these two 2-D gel systems, including a substantial number of basic proteins which had never been identified before. Proteins that were identified in both gel systems allowed us to cross-reference the gel patterns. Vector analysis of these cross...

  19. Molecular printboards as a general platform for protein immobilization: A supramolecular solution to nonspecific adsorption

    NARCIS (Netherlands)

    Ludden, M.J.W.; Mulder, A.; Tampe, Robert; Reinhoudt, David; Huskens, Jurriaan

    2007-01-01

    Be specific: A supramolecular adsorbate consisting of an adamantyl group (red) and an oligo(ethylene glycol) chain has been designed to prevent nonspecific protein adsorption at cyclodextrin molecular printboards. The adamantyl group allows specific and reversible interactions. Specific

  20. Decreased expression of extracellular matrix proteins and trophic factors in the amygdala complex of depressed mice after chronic immobilization stress

    Directory of Open Access Journals (Sweden)

    Jung Soonwoong

    2012-06-01

    Full Text Available Abstract Background The amygdala plays an essential role in controlling emotional behaviors and has numerous connections to other brain regions. The functional role of the amygdala has been highlighted by various studies of stress-induced behavioral changes. Here we investigated gene expression changes in the amygdala in the chronic immobilization stress (CIS-induced depression model. Results Eight genes were decreased in the amygdala of CIS mice, including genes for neurotrophic factors and extracellular matrix proteins. Among these, osteoglycin, fibromodulin, insulin-like growth factor 2 (Igf2, and insulin-like growth factor binding protein 2 (Igfbp2 were further analyzed for histological expression changes. The expression of osteoglycin and fibromodulin simultaneously decreased in the medial, basolateral, and central amygdala regions. However, Igf2 and Igfbp2 decreased specifically in the central nucleus of the amygdala. Interestingly, this decrease was found only in the amygdala of mice showing higher immobility, but not in mice displaying lower immobility, although the CIS regimen was the same for both groups. Conclusions These results suggest that the responsiveness of the amygdala may play a role in the sensitivity of CIS-induced behavioral changes in mice.

  1. PEG-Immobilized Keratin for Protein Drug Sequestration and pH-Mediated Delivery

    Directory of Open Access Journals (Sweden)

    Roche C. de Guzman

    2016-01-01

    Full Text Available Protein drugs like growth factors are promising therapeutics for damaged-tissue repair. Their local delivery often requires biomaterial carriers for achieving the therapeutic dose range while extending efficacy. In this study, polyethylene glycol (PEG and keratin were crosslinked and used as sponge-like scaffolds (KTN-PEG to absorb test proteins with different isoelectric points (pI: albumin (~5, hemoglobin (~7, and lysozyme (~11. The protein release kinetics was influenced by charge at physiological pH 7.4. The keratin network, with pI 5.3, electrostatically attracted lysozyme and repulsed albumin generating the release rate profile: albumin > hemoglobin > lysozyme. However, under acidic conditions (pH 4, all proteins including keratins were positively charged and consequently intermolecular repulsion altered the release hierarchy, now determined by size (MW diffusion: lysozyme (14 kDa > hemoglobin (64 kDa > albumin (66 kDa. Vascular endothelial growth factor C (VEGF-C, with properties comparable to lysozyme, was absorbed into the KTN-PEG scaffold. Endothelial cells cultured on this substrate had significantly larger numbers than on scaffolds without VEGF-C suggesting that the ionically bound and retained growth factor at neutral pH indirectly increased acute cell attachment and viability. PEG and keratin based sequestrations of proteins with basic pIs are therefore a feasible strategy with potential applications for selective biologics delivery.

  2. Lipid-Based Passivation in Nanofluidics

    Science.gov (United States)

    2012-01-01

    Stretching DNA in nanochannels is a useful tool for direct, visual studies of genomic DNA at the single molecule level. To facilitate the study of the interaction of linear DNA with proteins in nanochannels, we have implemented a highly effective passivation scheme based on lipid bilayers. We demonstrate virtually complete long-term passivation of nanochannel surfaces to a range of relevant reagents, including streptavidin-coated quantum dots, RecA proteins, and RecA–DNA complexes. We show that the performance of the lipid bilayer is significantly better than that of standard bovine serum albumin-based passivation. Finally, we show how the passivated devices allow us to monitor single DNA cleavage events during enzymatic degradation by DNase I. We expect that our approach will open up for detailed, systematic studies of a wide range of protein–DNA interactions with high spatial and temporal resolution. PMID:22432814

  3. Magnetic solid-phase extraction of protein with deep eutectic solvent immobilized magnetic graphene oxide nanoparticles.

    Science.gov (United States)

    Xu, Kaijia; Wang, Yuzhi; Ding, Xueqin; Huang, Yanhua; Li, Na; Wen, Qian

    2016-01-01

    As a new type of green solvent, four kinds of choline chloride (ChCl)-based deep eutectic solvents (DESs) have been synthesized, and then a core-shell structure magnetic graphene oxide (Fe3O4-NH2@GO) nanoparticles have been prepared and coated with the ChCl-based DESs. Magnetic solid-phase extraction (MSPE) based Fe3O4-NH2@GO@DES was studied for the first time for the extraction of proteins. The characteristic results of vibrating sample magnetometer (VSM), X-ray diffraction (XRD), Fourier transform infrared spectrometry (FT-IR), thermal gravimetric analysis (TGA) and field emission scanning electron microscopy (FESEM) indicated the successful preparation of Fe3O4-NH2@GO@DES. The concentrations of proteins in studies were determined by a UV-vis spectrophotometer. The advantages of Fe3O4-NH2@GO@DES in protein extraction were compared with Fe3O4-NH2@GO and Fe3O4-NH2, and Fe3O4-NH2@GO@ChCl-glycerol was selected as the suitable extraction solvent. The influence factors of the extraction process such as the pH value, the temperature, the extraction time, the concentration of protein and the amount of Fe3O4-NH2@GO@ChCl-glycerol were evaluated. Desorption experimental result showed 98.73% of BSA could be eluted from the solid extractant with 0.1 mol/L Na2HPO4 solution contained 1 mol/L NaCl. Besides, the conformation of BSA was not changed during the elution by the investigation of circular dichromism (CD) spectra. Furthermore, the analysis of real sample demonstrated that the prepared magnetic nanoparticles did have extraction ability on proteins in bovine whole blood. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Immobilization of L-glyceryl phosphorylcholine: isolation of phosphorylcholine-binding proteins from seminal plasma

    Czech Academy of Sciences Publication Activity Database

    Liberda, J.; Maňásková, Pavla; Švesták, M.; Jonáková, Věra; Tichá, M.

    2002-01-01

    Roč. 770, 1-2 (2002), s. 101-110 ISSN 0378-4347 R&D Projects: GA ČR GA303/99/0357; GA ČR GV524/96/K162 Institutional research plan: CEZ:AV0Z5052915; CEZ:MSM 113100001 Keywords : L-glyceryl phosphorylcholine * proteins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.913, year: 2002

  5. Immobilization of histidine-tagged proteins on monodisperse metallochelation liposomes: Preparation and study of their structure

    Czech Academy of Sciences Publication Activity Database

    Mašek, J.; Bartheldyová, E.; Korvasová, Z.; Škrabalová, J.; Koudelka, Š.; Kulich, P.; Kratochvílová, Irena; Miller, A. D.; Ledvina, Miroslav; Raška, M.; Turánek, J.

    2011-01-01

    Roč. 408, č. 1 (2011), s. 95-104 ISSN 0003-2697 R&D Projects: GA ČR(CZ) GAP304/10/1951 Institutional research plan: CEZ:AV0Z10100520; CEZ:AV0Z40550506 Keywords : liposome * proteoliposome * detergent removal method * recombinant protein * metallochelatation * AF microscopy * TEM * dynamic light scattering Subject RIV: CE - Biochemistry Impact factor: 2.996, year: 2011

  6. Photochemical immobilization of protein on the inner wall of a microchannel and Its application in a glucose sensor

    International Nuclear Information System (INIS)

    Nakajima, Hizuru; Ishino, Satomi; Masuda, Hironori; Nakagama, Tatsuro; Shimosaka, Takuya; Uchiyama, Katsumi

    2006-01-01

    A new protein immobilization technique has been developed for patterning enzymes in a specific position inside a microchannel. First, bovine serum albumin (BSA) was adsorbed onto the internal surface of a polydimethylsiloxane microchannel. The microchannel was then filled with the conjugate solution of a photoreactive cross-linker, 4-azido-2,3,5,6-tetrafluorobenzoic acid succinimidyl ester (ATFB-SE), and an enzyme, horseradish peroxidase (HRP). An irradiation by a He-Cd laser activated the azido group of the conjugates and these conjugates became covalently attached to the adsorbed BSA on the microchannel. The enzyme turnover was observed from only the HRP zone. This technique was successfully applied to the enzymatic glucose sensor. Glucose oxidase (GOD) and HRP were sequentially patterned in a single microchannel, i.e., the HRP zone was located downstream from the GOD zone. The calibration curve of a glucose standard solution was linear over the range of 0-128 μM with a correlation coefficient of 0.993. Compared to the traditional method using a 96-well microtiter plate, the present technique on the microchip shortened the reaction time from 30 min to 4.8 s, i.e., to 1/375

  7. State-of-the-art protein engineering approaches using biological macromolecules: A review from immobilization to implementation view point.

    Science.gov (United States)

    Bilal, Muhammad; Iqbal, Hafiz M N; Guo, Shuqi; Hu, Hongbo; Wang, Wei; Zhang, Xuehong

    2018-03-01

    Over the past years, technological and scientific advances have proven biocatalysis as a sustainable alternative than traditional chemical catalysis including organo- or metallocatalysis. In this context, immobilization based approaches represent simple but effective routes for engineering enzyme catalysts with higher activities than wild-type derivatives. Many enzymes including oxidoreductases have been engineered by rational and directed evolution, to realize the catalytic activity, enantioselectivity, and stability attributes which are essential for their biotechnological exploitation. Induce yet stable activity in enzyme catalysis offer new insights and motivation to engineer efficient catalysts for practical and commercial purposes. It has now become possible to envisage substrate accessibility to the catalytic site of the enzyme by current computational capabilities that reduce the experimental work related to the enzyme selection, screening, and engineering. Herein, state-of-the-art protein engineering approaches for improving enzymatic activities including chemical modification, directed evolution, and rational design or their combination methods are discussed. The emphasis is also given to the applications of the resulting tailored catalysts ranging from fine chemicals to novel pharmaceutical compounds that use biocatalysts as a vital step. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Protein covalent immobilization via its scarce thiol versus abundant amine groups: Effect on orientation, cell binding domain exposure and conformational lability.

    Science.gov (United States)

    Ba, O M; Hindie, M; Marmey, P; Gallet, O; Anselme, K; Ponche, A; Duncan, A C

    2015-10-01

    Quantity, orientation, conformation and covalent linkage of naturally cell adhesive proteins adsorbed or covalently linked to a surface, are known to influence the preservation of their subsequent long term cell adhesion properties and bioactivity. In the present work, we explore two different strategies for the covalent linking of plasma fibronectin (pFN) - used as a cell adhesive model protein, onto a polystyrene (PS) surface. One is aimed at tethering the protein to the surface in a semi-oriented fashion (via one of the 4 free thiol reactive groups on the protein) with a heterofunctional coupling agent (SSMPB method). The other aims to immobilize the protein in a more random fashion by reaction between the abundant pendant primary amine bearing amino acids of the pFN and activated carboxylic surface functions obtained after glutaric anhydride surface treatment (GA method). The overall goal will be to verify the hypothesis of a correlation between covalent immobilization of a model cell adhesive protein to a PS surface in a semi-oriented configuration (versus randomly oriented) with promotion of enhanced exposure of the protein's cell binding domain. This in turn would lead to enhanced cell adhesion. Ideally the goal is to elaborate substrates exhibiting a long term stable protein monolayer with preserved cell adhesive properties and bioactivity for biomaterial and/or cell adhesion commercial plate applications. However, the initial restrictive objective of this paper is to first quantitatively and qualitatively investigate the reversibly (merely adsorbed) versus covalently irreversibly bound protein to the surface after the immobilization procedure. Although immobilized surface amounts were similar (close to the monolayer range) for all immobilization approaches, covalent grafting showed improved retention and stronger "tethering" of the pFN protein to the surface (roughly 40%) after SDS rinsing compared to that for mere adsorption (0%) suggesting an added value

  9. Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry.

    Science.gov (United States)

    Siverino, Claudia; Tabisz, Barbara; Lühmann, Tessa; Meinel, Lorenz; Müller, Thomas; Walles, Heike; Nickel, Joachim

    2018-03-29

    Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

  10. Desorption of Lipases Immobilized on Octyl-Agarose Beads and Coated with Ionic Polymers after Thermal Inactivation. Stronger Adsorption of Polymers/Unfolded Protein Composites

    Directory of Open Access Journals (Sweden)

    Jose J. Virgen-Ortíz

    2017-01-01

    Full Text Available Lipases from Candida antarctica (isoform B and Rhizomucor miehei (CALB and RML have been immobilized on octyl-agarose (OC and further coated with polyethylenimine (PEI and dextran sulfate (DS. The enzymes just immobilized on OC supports could be easily released from the support using 2% SDS at pH 7, both intact or after thermal inactivation (in fact, after inactivation most enzyme molecules were already desorbed. The coating with PEI and DS greatly reduced the enzyme release during thermal inactivation and improved enzyme stability. However, using OC-CALB/RML-PEI-DS, the full release of the immobilized enzyme to reuse the support required more drastic conditions: a pH value of 3, a buffer concentration over 2 M, and temperatures above 45 °C. However, even these conditions were not able to fully release the thermally inactivated enzyme molecules from the support, being necessary to increase the buffer concentration to 4 M sodium phosphate and decrease the pH to 2.5. The formation of unfolded protein/polymers composites seems to be responsible for this strong interaction between the octyl and some anionic groups of OC supports. The support could be reused five cycles using these conditions with similar loading capacity of the support and stability of the immobilized enzyme.

  11. Immobilization of cross linked Col-I–OPN bone matrix protein on aminolysed PCL surfaces enhances initial biocompatibility of human adipogenic mesenchymal stem cells (hADMSC)

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young-Hee; Jyoti, Md. Anirban; Song, Ho-Yeon, E-mail: songmic@sch.ac.kr

    2014-06-01

    In bone tissue engineering surface modification is considered as one of the important ways of fabricating successful biocompatible material. Addition of biologically active functionality on the surfaces has been tried for improving the overall biocompatibility of the system. In this study poly-ε-caprolactone film surfaces have been modified through aminolysis and immobilization process. Collagen type I (COL-I) and osteopontin (OPN), which play an important role in osteogenesis, was immobilized onto PCL films followed by aminolysis treatment using 1,6-hexanediamine. Characterization of animolysed and immobilized surfaces were done by a number techniques using scanning electron microscopy (SEM), FT-IR, XPS, ninhydrin staining, SDS-PAGE and confocal microscopy and compared between the modified and un-modified surfaces. Results of the successive experiments showed that aminolysis treatment was homogeneously achieved which helped to entrap or immobilize Col-I–OPN proteins on surfaces of PCL film. In vitro studies with human adipogenic mesenchymal stem cells (hADMSC) also confirmed the attachment and proliferation of cells was better in modified PCL surfaces than the unmodified surfaces. SEM, confocal microscopy and MTT assay showed a significant increase in cell spreading, attachment and proliferations on the biofunctionalized surfaces compared to the unmodified PCL surfaces at all-time points indicating the success of surface biofunctionalization.

  12. Direct Profiling the Post-Translational Modification Codes of a Single Protein Immobilized on a Surface Using Cu-free Click Chemistry.

    Science.gov (United States)

    Kim, Kyung Lock; Park, Kyeng Min; Murray, James; Kim, Kimoon; Ryu, Sung Ho

    2018-05-23

    Combinatorial post-translational modifications (PTMs), which can serve as dynamic "molecular barcodes", have been proposed to regulate distinct protein functions. However, studies of combinatorial PTMs on single protein molecules have been hindered by a lack of suitable analytical methods. Here, we describe erasable single-molecule blotting (eSiMBlot) for combinatorial PTM profiling. This assay is performed in a highly multiplexed manner and leverages the benefits of covalent protein immobilization, cyclic probing with different antibodies, and single molecule fluorescence imaging. Especially, facile and efficient covalent immobilization on a surface using Cu-free click chemistry permits multiple rounds (>10) of antibody erasing/reprobing without loss of antigenicity. Moreover, cumulative detection of coregistered multiple data sets for immobilized single-epitope molecules, such as HA peptide, can be used to increase the antibody detection rate. Finally, eSiMBlot enables direct visualization and quantitative profiling of combinatorial PTM codes at the single-molecule level, as we demonstrate by revealing the novel phospho-codes of ligand-induced epidermal growth factor receptor. Thus, eSiMBlot provides an unprecedentedly simple, rapid, and versatile platform for analyzing the vast number of combinatorial PTMs in biological pathways.

  13. Immobilization of Rhizobial Exopolysaccharides and Nod Factors Provides a Novel Platform for Interaction with Proteins

    DEFF Research Database (Denmark)

    Hjuler, Christian Toftegaard

    Legumes are plants essential to human nutrition, because of their seeds that include beans, lentils and peas. In academia, legumes are especially studied due to their auxiliary ability to fix nitrogen, which is derived from a symbiosis with rhizobial bacteria. This thesis seeks to expand the curr......Legumes are plants essential to human nutrition, because of their seeds that include beans, lentils and peas. In academia, legumes are especially studied due to their auxiliary ability to fix nitrogen, which is derived from a symbiosis with rhizobial bacteria. This thesis seeks to expand...... genetic studies performed by Kawaharada et al. at Aarhus University. As LYS3 was confirmed to be a receptor for exopolysaccharides this led to the protein being renamed ExoPolysaccharide Receptor 3 (EPR3...

  14. Enhanced bioavailability of nerve growth factor with phytantriol lipid-based crystalline nanoparticles in cochlea

    Directory of Open Access Journals (Sweden)

    Bu M

    2015-11-01

    Full Text Available Meng Bu,1,2 Jingling Tang,3 Yinghui Wei,4 Yanhui Sun,1 Xinyu Wang,1 Linhua Wu,2 Hongzhuo Liu1 1School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China; 2Department of Pharmacy, the Second Affiliated Hospital, 3School of Pharmacy, Harbin Medical University, Harbin, People’s Republic of China; 4College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China Purpose: Supplementation of exogenous nerve growth factor (NGF into the cochlea of deafened animals rescues spiral ganglion cells from degeneration. However, a safe and potent delivery of therapeutic proteins, such as NGF, to spiral ganglion cells remains one of the greatest challenges. This study presents the development of self-assembled cubic lipid-based crystalline nanoparticles to enhance inner ear bioavailability of bioactive NGF via a round window membrane route.Methods: A novel nanocarrier-entrapped NGF was developed based on phytantriol by a liquid precursor dilution, with Pluronic® F127 and propylene glycol as the surfactant and solubilizer, respectively. Upon dilution of the liquid lipid precursors, monodispersed submicron-sized particles with a slight negative charge formed spontaneously.Results: Biological activity of entrapped NGF was assessed using pheochromocytoma cells with NGF-loaded reservoirs to induce significant neuronal outgrowth, similar to that seen in free NGF-treated controls. Finally, a 3.28-fold increase in inner ear bioavailability was observed after administration of phytantriol lipid-based crystalline nanoparticles as compared to free drug, contributing to an enhanced drug permeability of the round window membrane. Conclusion: Data presented here demonstrate the potential of lipid-based crystalline nanoparticles to improve the outcomes of patients bearing cochlear implants. Keywords: nerve growth factor, lipid-based crystalline nanoparticles, PC12 cells, inner ear drug

  15. Heterogeneous electron transfer of a two-centered heme protein: redox and electrocatalytic properties of surface-immobilized cytochrome C(4).

    Science.gov (United States)

    Monari, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; Di Rocco, Giulia; Martini, Laura; Ranieri, Antonio; Sola, Marco

    2009-10-15

    The recombinant diheme cytochrome c(4) from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 and its Met64Ala and Met164Ala variants, which feature a hydroxide ion axially bound to the heme iron at the N- and C-terminal domains, respectively, were found to exchange electrons efficiently with a gold electrode coated with a SAM of 11-mercapto-1-undecanoic acid. The mutation-induced removal of the redox equivalence of the two heme groups and changes in the net charge of the protein lobes yield two-centered protein systems with unprecedented properties in the electrode-immobilized state. The heterogeneous and intraheme electron transfer processes were characterized for these species in which the high- and low-potential heme groups are swapped over in the bilobal protein framework and experience a constrained (M64A) and unconstrained (M164A) orientation toward the electrode. The reduction thermodynamics for the native and mutated hemes were measured for the first time for a diheme cytochrome c. In the diffusing regime, they reproduce closely those for the corresponding centers in single-heme class-I cytochromes c, despite the low sequence identity. Larger differences are observed in the thermodynamics of the immobilized species and in the heterogeneous electron transfer rate constants. T-dependent kinetic measurements show that the proteins are positioned approximately 7 A from the HOOC-terminated SAM-coated electrode. Protein-electrode orientation and efficient intraheme ET enable the His,OH(-)-ligated heme A of the immobilized Met64Ala variant to carry out the reductive electrocatalysis of molecular oxygen. This system therefore constitutes a novel two-centered heme-based biocatalytic interface to be exploited for "third-generation" amperometric biosensing.

  16. Microorganism immobilization

    Science.gov (United States)

    Compere, Alicia L.; Griffith, William L.

    1981-01-01

    Live metabolically active microorganisms are immobilized on a solid support by contacting particles of aggregate material with a water dispersible polyelectrolyte such as gelatin, crosslinking the polyelectrolyte by reacting it with a crosslinking agent such as glutaraldehyde to provide a crosslinked coating on the particles of aggregate material, contacting the coated particles with live microorganisms and incubating the microorganisms in contact with the crosslinked coating to provide a coating of metabolically active microorganisms. The immobilized microorganisms have continued growth and reproduction functions.

  17. Recent developments in oral lipid-based drug delivery

    DEFF Research Database (Denmark)

    Thomas, N.; Rades, T.; Müllertz, A.

    2013-01-01

    The increasing number of poorly water-soluble drugs in development in the pharmaceutical industry has sparked interest in novel drug delivery options such as lipid-based drug delivery systems (LbDDS). Several LbDDS have been marketed successfully and have shown superior and more reliable...... bioavailability compared to conventional formulations. However, some reluctance in the broader application of LbDDS still appears, despite the growing commercial interest in lipids as a drug delivery platform. This reluctance might at least in part be related to the complexity associated with the development...... and characterization of LbDDS. In particular, the lack of standardized test protocols can be identified as the major obstacles for the broader application of LbDDS. This review seeks to summarize recent approaches in the field of lipid-based drug delivery that try to elucidate some critical steps in their development...

  18. Penetrable silica microspheres for immobilization of bovine serum albumin and their application to the study of the interaction between imatinib mesylate and protein by frontal affinity chromatography.

    Science.gov (United States)

    Ma, Liyun; Li, Jing; Zhao, Juan; Liao, Han; Xu, Li; Shi, Zhi-guo

    2016-01-01

    In the current study, novel featured silica, named penetrable silica, simultaneously containing macropores and mesopores, was immobilized with bovine serum albumin (BSA) via Schiff base method. The obtained BSA-SiO2 was employed as the high-performance liquid chromatographic (HPLC) stationary phase. Firstly, D- and L-tryptophan were used as probes to investigate the chiral separation ability of the BSA-SiO2 stationary phase. An excellent enantioseparation factor was obtained up to 4.3 with acceptable stability within at least 1 month. Next, the BSA-SiO2 stationary phase was applied to study the interaction between imatinib mesylate (IM) and BSA by frontal affinity chromatography. A single type of binding site was found for IM with the immobilized BSA, and the hydrogen-bonding and van der Waals interactions were expected to be contributing interactions based on the thermodynamic studies, and this was a spontaneous process. Compared to the traditional silica for HPLC stationary phase, the proposed penetrable silica microsphere possessed a larger capacity to bond more BSA, minimizing column overloading effects and enhancing enantioseparation ability. In addition, the lower running column back pressure and fast mass transfer were meaningful for the column stability and lifetime. It was a good substrate to immobilize biomolecules for fast chiral resolution and screening drug-protein interactions.

  19. Electrochemical investigations of the interaction of C-reactive protein (CRP) with a CRP antibody chemically immobilized on a gold surface

    International Nuclear Information System (INIS)

    Hennessey, Hooman; Afara, Nadia; Omanovic, Sasha; Padjen, Ante L.

    2009-01-01

    A possibility of using a range of dc and ac electrochemical techniques to probe associative interactions of C-reactive protein (CRP) with CRP antibody (aCRP) immobilized on a gold electrode surface was investigated. It was demonstrated that the investigated electrochemical techniques can be used efficiently to probe these interactions over a wide CRP concentration range, from 1.15 x 10 -5 to 1.15 mg L -1 . The measured sensitivity of the techniques is in the following decreasing order: differential pulse voltammetry, charge-transfer resistance obtained from electrochemical impedance spectroscopy (EIS), cyclic voltammetry, chronoamperometry, and double-layer capacitance deduced from EIS measurements which gave the poorest sensitivity. Measurements of kinetic parameters demonstrated that the associative interactions of CRP with the immobilized aCRP reached quasi-equilibrium after 20-30 min. The kinetics of these interactions was modeled successfully using a two-step kinetic model. In this model, the first step represents reversible CRP-aCRP associative-dissociative interactions, while the second step represents the irreversible transformation of the bound CRP into a thermodynamically stable configuration. It was demonstrated that the thermodynamically stable configuration of CRP starts prevailing after 7 min of interaction of CRP with the immobilized aCRP.

  20. Lipid Based Nanosystems for Curcumin: Past, Present and Future.

    Science.gov (United States)

    Nayak, Aditya P; Mills, Tom; Norton, Ian

    2016-01-01

    Curcumin is one of the principle bioactive compounds used in the ayurvedic medicine system that has the history of over 5000 years for human use. Curcumin an "Indian Gold" is used to treat simple ailments like the common cold to severe life threatening diseases like cancer, and HIV. Though its contribution is immense for the health protection and disease prevention, its clinical use is limited due to its susceptible nature to alkaline pH, high temperature, presence of oxygen and light. Hence it becomes extremely difficult to maintain its bioactivity during processing, storage and consumption. Recent advancements in the application of nanotechnology to curcumin offer an opportunity to enhance its stability, bioactivity and to overcome its pharmacokinetic mismatch. This in turn helps to bridge the gaps that exist between its bench top research data to its clinical findings. Among the various types of nano/micro delivery systems, lipid based delivery systems are well studied and are the best suited delivery systems to enhance the stability and pharmacokinetic profile of curcumin both for pharma and the food application. In the current review, effort will be made to recapitulate the work done in the past to use lipid based delivery systems (liposomes, solid lipid nanoparticles, and emulsions) to enhance the application of curcumin for health promotion and disease prevention. Further, future prospects for the utilization of these lipid-based delivery systems will be discussed in detail.

  1. Recombinant immobilized rhizopuspepsin as a new tool for protein digestion in hydrogen/deuterium exchange mass spectrometry

    Czech Academy of Sciences Publication Activity Database

    Rey, M.; Man, Petr; Brandolin, G.; Forest, E.; Pelosi, L.

    2009-01-01

    Roč. 23, č. 21 (2009), s. 3431-3438 ISSN 0951-4198 Institutional research plan: CEZ:AV0Z50200510 Keywords : mass spectrometry * recombinant immobilized rhizopuspepsin * protease Subject RIV: CE - Biochemistry Impact factor: 2.695, year: 2009

  2. RAD18 and associated proteins are immobilized in nuclear foci in human cells entering S-phase with ultraviolet light-induced damage

    International Nuclear Information System (INIS)

    Watson, Nicholas B.; Nelson, Eric; Digman, Michelle; Thornburg, Joshua A.; Alphenaar, Bruce W.; McGregor, W. Glenn

    2008-01-01

    Proteins required for translesion DNA synthesis localize in nuclear foci of cells with replication-blocking lesions. The dynamics of this process were examined in human cells with fluorescence-based biophysical techniques. Photobleaching recovery and raster image correlation spectroscopy experiments indicated that involvement in the nuclear foci reduced the movement of RAD18 from diffusion-controlled to virtual immobility. Examination of the mobility of REV1 indicated that it is similarly immobilized when it is observed in nuclear foci. Reducing the level of RAD18 greatly reduced the focal accumulation of REV1 and reduced UV mutagenesis to background frequencies. Fluorescence lifetime measurements indicated that RAD18 and RAD6A or polη only transferred resonance energy when these proteins colocalized in damage-induced nuclear foci, indicating a close physical association only within such foci. Our data support a model in which RAD18 within damage-induced nuclear foci is immobilized and is required for recruitment of Y-family DNA polymerases and subsequent mutagenesis. In the absence of damage these proteins are not physically associated within the nucleoplasm

  3. Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

    International Nuclear Information System (INIS)

    Moaddel, Ruin; Wainer, Irving W.

    2006-01-01

    Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K d values) and non-linear chromatography can be used to assess the association (k on ) and dissociation (k off ) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein

  4. Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

    Energy Technology Data Exchange (ETDEWEB)

    Moaddel, Ruin [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States); Wainer, Irving W. [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States)]. E-mail: Wainerir@grc.nia.nih.gov

    2006-03-30

    Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K {sub d} values) and non-linear chromatography can be used to assess the association (k {sub on}) and dissociation (k {sub off}) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein.

  5. Biobased, Internally pH-Sensitive Materials: Immobilized Yellow Fluorescent Protein as an Optical Sensor for Spatiotemporal Mapping of pH Inside Porous Matrices.

    Science.gov (United States)

    Consolati, Tanja; Bolivar, Juan M; Petrasek, Zdenek; Berenguer, Jose; Hidalgo, Aurelio; Guisán, Jose M; Nidetzky, Bernd

    2018-02-28

    The pH is fundamental to biological function and its measurement therefore crucial across all biosciences. Unlike homogenous bulk solution, solids often feature internal pH gradients due to partition effects and confined biochemical reactions. Thus, a full spatiotemporal mapping for pH characterization in solid materials with biological systems embedded in them is essential. In here, therefore, a fully biocompatible methodology for real-time optical sensing of pH within porous materials is presented. A genetically encoded ratiometric pH sensor, the enhanced superfolder yellow fluorescent protein (sYFP), is used to functionalize the internal surface of different materials, including natural and synthetic organic polymers as well as silica frameworks. By using controlled, tailor-made immobilization, sYFP is homogenously distributed within these materials and so enables, via self-referenced imaging analysis, pH measurements in high accuracy and with useful spatiotemporal resolution. Evolution of internal pH is monitored in consequence of a proton-releasing enzymatic reaction, the hydrolysis of penicillin by a penicillin acylase, taking place in solution or confined to the solid surface of the porous matrix. Unlike optochemical pH sensors, which often interfere with biological function, labeling with sYFP enables pH sensing without altering the immobilized enzyme's properties in any of the materials used. Fast response of sYFP to pH change permits evaluation of biochemical kinetics within the solid materials. Thus, pH sensing based on immobilized sYFP represents a broadly applicable technique to the study of biology confined to the internally heterogeneous environment of solid matrices.

  6. Immobilized Cytochrome P450 2C9 (CYP2C9): Applications for Metabolite Generation, Monitoring Protein-Protein Interactions, and Improving In-vivo Predictions Using Enhanced In-vitro Models

    Science.gov (United States)

    Wollenberg, Lance A.

    Cytochrome P450 (P450) enzymes are a family of oxoferroreductase enzymes containing a heme moiety and are well known to be involved in the metabolism of a wide variety of endogenous and xenobiotic materials. It is estimated that roughly 75% of all pharmaceutical compounds are metabolized by these enzymes. Traditional reconstituted in-vitro incubation studies using recombinant P450 enzymes are often used to predict in-vivo kinetic parameters of a drug early in development. However, in many cases, these reconstituted incubations are prone to aggregation which has been shown to affect the catalytic activity of an enzyme. Moreover, the presence of other isoforms of P450 enzymes present in a metabolic incubation, as is the case with microsomal systems, may affect the catalytic activity of an enzyme through isoform-specific protein-protein interactions. Both of these effects may result in inaccurate prediction of in-vivo drug metabolism using in-vitro experiments. Here we described the development of immobilized P450 constructs designed to elucidate the effects of aggregation and protein-protein interactions between P450 isoforms on catalytic activities. The long term objective of this project is to develop a system to control the oligomeric state of Cytochrome P450 enzymes to accurately elucidate discrepancies between in vitro reconstituted systems and actual in vivo drug metabolism for the precise prediction of metabolic activity. This approach will serve as a system to better draw correlations between in-vivo and in-vitro drug metabolism data. The central hypothesis is that Cytochrome P450 enzymes catalytic activity can be altered by protein-protein interactions occurring between Cytochrome P450 enzymes involved in drug metabolism, and is dependent on varying states of protein aggregation. This dissertation explains the details of the construction and characterization of a nanostructure device designed to control the state of aggregation of a P450 enzyme. Moreover

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

    Science.gov (United States)

    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.

  8. Acetylcholinesterase immobilized capillary reactors coupled to protein coated magnetic beads: A new tool for plant extract ligand screening

    OpenAIRE

    Vanzolini, Kenia Lourenço; Jiang, Zhengjin; Zhang, Xiaoqi; Vieira, Lucas Campos Curcino; Corrêa, Arlene Gonçalvez; Cardoso, Carmen Lucia; Cass, Quezia Bezerra; Moaddel, Ruin

    2013-01-01

    The use of immobilized capillary enzyme reactors (ICERs) and enzymes coated to magnetic beads ((NT or CT)-MB) for ligand screening has been adopted as a new technique of high throughput screening (HTS). In this work the selected target was the enzyme acetylcholinesterase (AChE), which acts on the central nervous system and is a validated target for the treatment of Alzheimer’s disease, as well as for new insecticides. A new approach for the screening of plant extracts was developed based on t...

  9. Two-step purification of His-tagged Nef protein in native condition using heparin and immobilized metal ion affinity chromatographies.

    Science.gov (United States)

    Finzi, Andrés; Cloutier, Jonathan; Cohen, Eric A

    2003-07-01

    The Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.

  10. Enzymes as Biocatalysts for Lipid-based Bioproducts Processing

    DEFF Research Database (Denmark)

    Cheong, Ling-Zhi; Guo, Zheng; Fedosov, Sergey

    2012-01-01

    Bioproducts are materials, chemicals and energy derived from renewable biological resources such as agriculture, forestry, and biologically-derived wastes. To date, the use of enzymes as biocatalysts for lipid-based bioproducts processing has shown marked increase. This is mainly due to the fact...... that cost benefit derived from enzymatic processing such as enzyme specificity, higher product purity and lesser or none toxic waste disposal has surpassed the cost of biocatalysts itself. This chapter provided insights into distinct enzymes characteristics essential in industrial processing especially...... enzymes kinetics. Understanding of enzyme kinetics is important especially in designing efficient reaction set-ups including type of bioreactors, reaction conditions and reusability of biocatalysts to ensure efficient running cost. A brief review of state-of-the-art in industrial applications of enzymes...

  11. Acetylcholinesterase immobilized capillary reactors coupled to protein coated magnetic beads: A new tool for plant extract ligand screening

    Science.gov (United States)

    Vanzolini, Kenia Lourenço; Jiang, Zhengjin; Zhang, Xiaoqi; Vieira, Lucas Campos Curcino; Corrêa, Arlene Gonçalvez; Cardoso, Carmen Lucia; Cass, Quezia Bezerra; Moaddel, Ruin

    2013-01-01

    The use of immobilized capillary enzyme reactors (ICERs) and enzymes coated to magnetic beads ((NT or CT)-MB) for ligand screening has been adopted as a new technique of high throughput screening (HTS). In this work the selected target was the enzyme acetylcholinesterase (AChE), which acts on the central nervous system and is a validated target for the treatment of Alzheimer’s disease, as well as for new insecticides. A new approach for the screening of plant extracts was developed based on the ligand fishing experiments and zonal chromatography. For that, the magnetic beads were used for the ligand fishing experiments and capillary bioreactors for the activity assays. The latter was employed also under non-linear conditions to determine the affinity constants of known ligands, for the first time, as well as for the active fished ligand. PMID:24148457

  12. Transforming lipid-based oral drug delivery systems into solid dosage forms: an overview of solid carriers, physicochemical properties, and biopharmaceutical performance.

    Science.gov (United States)

    Tan, Angel; Rao, Shasha; Prestidge, Clive A

    2013-12-01

    The diversity of lipid excipients available commercially has enabled versatile formulation design of lipid-based drug delivery systems for enhancing the oral absorption of poorly water-soluble drugs, such as emulsions, microemulsions, micelles, liposomes, niosomes and various self-emulsifying systems. The transformation of liquid lipid-based systems into solid dosage forms has been investigated for several decades, and has recently become a core subject of pharmaceutical research as solidification is regarded as viable means for stabilising lipid colloidal systems while eliminating stringent processing requirements associated with liquid systems. This review describes the types of pharmaceutical grade excipients (silica nanoparticle/microparticle, polysaccharide, polymer and protein-based materials) used as solid carriers and the current state of knowledge on the liquid-to-solid conversion approaches. Details are primarily focused on the solid-state physicochemical properties and redispersion capacity of various dry lipid-based formulations, and how these relate to the in vitro drug release and solubilisation, lipid carrier digestion and cell permeation performances. Numerous in vivo proof-of-concept studies are presented to highlight the viability of these dry lipid-based formulations. This review is significant in directing future research work in fostering translation of dry lipid-based formulations into clinical applications.

  13. Charge stabilization by reaction center protein immobilized to carbon nanotubes functionalized by amine groups and poly(3-thiophene acetic acid) conducting polymer

    Energy Technology Data Exchange (ETDEWEB)

    Szabo, T.; Magyar, M.; Nagy, L. [Department of Medical Physics and Informatics, University of Szeged, H-6720 Szeged (Hungary); Nemeth, Z.; Hernadi, K. [Department of Applied and Environmental Chemistry, University of Szeged, H-6720 Szeged (Hungary); Endrodi, B.; Bencsik, G.; Visy, Cs. [Department of Physical Chemistry and Materials Science, University of Szeged, H-6720 Szeged (Hungary); Horvath, E.; Magrez, A.; Forro, L. [Institute of Physics of Complex Matter, Ecole Polytechnique Federale de Lausanne, CH-1015 Lausanne (Switzerland)

    2012-12-15

    A large number of studies have indicated recently that photosynthetic reaction center proteins (RC) bind successfully to nanostructures and their functional activity is largely retained. The major goal of current research is to find the most efficient systems and conditions for the photoelectric energy conversion and for the stability of this bio-nanocomposite. In our studies, we immobilized the RC protein on multiwalled carbon nanotubes (MWNT) through specific chemical binding to amine functional groups and through conducting polymer (poly(3-thiophene acetic acid), PTAA). Both structural (TEM, AFM) and functional (absorption change and conductivity) measurements has shown that RCs could be bound effectively to functionalized CNTs. The kinetics of the light induced absorption change indicated that RCs were still active in the composite and there was an interaction between the protein cofactors and the CNTs. The light generated photocurrent was measured in an electrochemical cell with transparent CNT electrode designed specially for this experiment. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  14. Effect of Calcium β-Hydroxy-β-Methylbutyrate (CaHMB), Vitamin D, and Protein Supplementation on Postoperative Immobilization in Malnourished Older Adult Patients With Hip Fracture: A Randomized Controlled Study.

    Science.gov (United States)

    Ekinci, Osman; Yanık, Serhat; Terzioğlu Bebitoğlu, Berna; Yılmaz Akyüz, Elvan; Dokuyucu, Ayfer; Erdem, Şevki

    2016-12-01

    Nutrition support in orthopedic patients with malnutrition shortens the immobilization period. The efficacy of calcium β-hydroxy-β-methylbutyrate (CaHMB), vitamin D, and protein intake on bone structure is studied and well known; however, there is no evidence supporting the effect of combined use in orthopedic conditions. We investigated the effects of CaHMB, vitamin D, and protein supplementation on wound healing, immobilization period, muscle strength, and laboratory parameters. This randomized controlled study included 75 older female patients with a hip fracture admitted to orthopedic clinics. The control group received standard postoperative nutrition. The study group received an enteral product containing 3 g CaHMB, 1000 IU vitamin D, and 36 g protein, in addition to standard postoperative nutrition. Anthropometric, laboratory, wound-healing, immobilization period, and muscle strength assessments were evaluated preoperatively and on postoperative days 15 and 30. Wound-healing period was significantly shorter in the CaHMB/vitamin D/protein group than in the control group ( P patients in the CaHMB/vitamin D/protein group who were mobile on days 15 and 30 (81.3%) was significantly higher than patients in the control group, who were mobile on days 15 and 30 (26.7%) ( P = .001). Muscle strength on day 30 was significantly higher in the CaHMB/vitamin D/protein group vs the control group. Nutrition of elderly patients with a CaHMB/vitamin D/protein combination led to acceleration of wound healing, shortening of immobilization period, and increased muscle strength without changing body mass index. It also reduced dependence to bed and related complications after an orthopedic operation.

  15. Drug loading to lipid-based cationic nanoparticles

    International Nuclear Information System (INIS)

    Cavalcanti, Leide P.; Konovalov, Oleg; Torriani, Iris L.; Haas, Heinrich

    2005-01-01

    Lipid-based cationic nanoparticles are a new promising option for tumor therapy, because they display enhanced binding and uptake at the neo-angiogenic endothelial cells, which a tumor needs for its nutrition and growth. By loading suitable cytotoxic compounds to the cationic carrier, the tumor endothelial and consequently also the tumor itself can be destroyed. For the development of such novel anti-tumor agents, the control of drug loading and drug release from the carrier matrix is essential. We have studied the incorporation of the hydrophobic anti-cancer agent Paclitaxel (PXL) into a variety of lipid matrices by X-Ray reflectivity measurements. Liposome suspensions from cationic and zwitterionic lipids, comprising different molar fractions of Paclitaxel, were deposited on planar glass substrates. After drying at controlled humidity, well ordered, oriented multilayer stacks were obtained, as proven by the presence of bilayer Bragg peaks to several orders in the reflectivity curves. The presence of the drug induced a decrease of the lipid bilayer spacing, and with an excess of drug, also Bragg peaks of drug crystals could be observed. From the results, insight into the solubility of Paclitaxel in the model membranes was obtained and a structural model of the organization of the drug in the membrane was derived. Results from subsequent pressure/area-isotherm and grazing incidence diffraction (GID) measurements performed with drug/lipid Langmuir monolayers were in accordance with these conjectures

  16. Immobilization/Stabilization of Ficin Extract on Glutaraldehyde-Activated Agarose Beads. Variables That Control the Final Stability and Activity in Protein Hydrolyses

    Directory of Open Access Journals (Sweden)

    El-Hocine Siar

    2018-04-01

    Full Text Available Ficin extract has been immobilized on different 4% aminated-agarose beads. Using just ion exchange, immobilization yield was poor and expressed activity did not surpass 10% of the offered enzyme, with no significant effects on enzyme stability. The treatment with glutaraldehyde of this ionically exchanged enzyme produced an almost full enzyme inactivation. Using aminated supports activated with glutaraldehyde, immobilization was optimal at pH 7 (at pH 5 immobilization yield was 80%, while at pH 9, the immobilized enzyme became inactivated. At pH 7, full immobilization was accomplished maintaining 40% activity versus a small synthetic substrate and 30% versus casein. Ficin stabilization upon immobilization could be observed but it depended on the inactivation pH and the substrate employed, suggesting the complexity of the mechanism of inactivation of the immobilized enzyme. The maximum enzyme loading on the support was determined to be around 70 mg/g. The loading has no significant effect on the enzyme stability or enzyme activity using the synthetic substrate but it had a significant effect on the activity using casein; the biocatalysts activity greatly decreased using more than 30 mg/g, suggesting that the near presence of other immobilized enzyme molecules may generate some steric hindrances for the casein hydrolysis.

  17. The effect of bound to dialdehudecellulose protein concentration on the activity of immobilized trypsin after γ-irradiation and in process of storage

    International Nuclear Information System (INIS)

    Belov, A.A.; Ryl'tsev, V.V.; Ignatyuk, T.E.; Filatov, V.N.

    1994-01-01

    It is found the complex effect of the bound enzyme concentration on the proteolytic activity of trypsin immobilized to dialdehydecellulose (preriodate oxidation) after γ-irradiation and in process of storage. It is shown the occurance of three stages of immobilized enzyme inactivation in process of immobilization and storage. The velocity of inactivation did not depend on bound trypsin concentration. The ratio of proteolytic activity of samples before and after γ-irradiation was increased with the increase of immobilized to carrier enzyme concentration and was not change (in range of experiment error) in process of storage. The results were compared with that of cryctlline trypsin

  18. Computer-aided design of bromelain and papain covalent immobilization

    OpenAIRE

    Cutiño-Avila, Bessy; Gil Pradas, Dayrom; Aragón Abreu, Carlos; Fernández Marrero, Yuniel; Hernández de la Torre, Martha; Salas Sarduy, Emir; Chávez Planes, María de los Ángeles; Guisán Seijas, José Manuel; Díaz Brito, Joaquín; del Monte-Martínez, Alberto

    2014-01-01

    Enzymes as immobilized derivatives have been widely used in Food, Agrochemical, Pharmaceutical and Biotechnological industries. Protein immobilization is probably the most used technology to improve the operational stability of these molecules. Bromelain (Ananas comosus) and papain (Carica papaya) are cystein proteases extensively used as immobilized biocatalyst with several applications in therapeutics, racemic mixtures resolution, affinity chromatography and others industrial scenarios. The...

  19. Immobilization of bacterial S-layer proteins from Caulobacter crescentus on iron oxide-based nanocomposite: synthesis and spectroscopic characterization of zincite-coated Fe₂O₃ nanoparticles.

    Science.gov (United States)

    Habibi, Neda

    2014-05-05

    Zinc oxide was coated on Fe2O3 nanoparticles using sol-gel spin-coating. Caulobacter crescentus have a crystalline surface layer (S-layer), which consist of one protein or glycoprotein species. The immobilization of bacterial S-layers obtained from C. crescentus on zincite-coated nanoparticles of iron oxide was investigated. The SDS PAGE results of S-layers isolated from C. crescentus showed the weight of 50 KDa. Nanoparticles of the Fe2O3 and zinc oxide were synthesized by a sol-gel technique. Fe2O3 nanoparticles with an average size of 50 nm were successfully prepared by the proper deposition of zinc oxide onto iron oxide nanoparticles surface annealed at 450 °C. The samples were characterized by field-emission scanning electron microscope (FESEM), atomic force microscopy (AFM), powder X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FT-IR). Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Relevance of Lipid-Based Products in the Management of Dry Eye Disease.

    Science.gov (United States)

    Garrigue, Jean-Sébastien; Amrane, Mourad; Faure, Marie-Odile; Holopainen, Juha M; Tong, Louis

    2017-11-01

    Components of the ocular surface synergistically contribute to maintaining and protecting a smooth refractive layer to facilitate the optimal transmission of light. At the air-water interface, the tear film lipid layer (TFLL), a mixture of lipids and proteins, plays a key role in tear surface tension and is important for the physiological hydration of the ocular surface and for ocular homeostasis. Alterations in tear fluid rheology, differences in lipid composition, or downregulation of specific tear proteins are found in most types of ocular surface disease, including dry eye disease (DED). Artificial tears have long been a first line of treatment in DED and aim to replace or supplement tears. More recently, lipid-containing eye drops have been developed to more closely mimic the combination of aqueous and lipid layers of the TFLL. Over the last 2 decades, our understanding of the nature and importance of lipids in the tear film in health and disease has increased substantially. The aim of this article is to provide a brief overview of our current understanding of tear film properties and review the effectiveness of lipid-based products in the treatment of DED. Liposome lid sprays, emulsion eye drops, and other lipid-containing formulations are discussed.

  1. Impact of change of matrix crystallinity and polymorphism on ovalbumin release from lipid-based implants.

    Science.gov (United States)

    Duque, Luisa; Körber, Martin; Bodmeier, Roland

    2018-05-30

    The objectives of this study were to prepare lipid-based implants by hot melt extrusion (HME) for the prolonged release of ovalbumin (OVA), and to relate protein release to crystallinity and polymorphic changes of the lipid matrix. Two lipids, glycerol tristearate and hydrogenated palm oil, with different composition and degree of crystallinity were studied. Solid OVA was dispersed within the lipid matrixes, which preserved its stability during extrusion. This was partially attributed to a protective effect of the lipidic matrix. The incorporation of OVA decreased the mechanical strength of the implants prepared with the more crystalline matrix, glycerol tristearate, whereas it remained comparable for the hydrogenated palm oil because of stronger physical and non-covalent interactions between the protein and this lipid. This was also the reason for the faster release of OVA from the glycerol tristearate matrix when compared to the hydrogenated palm oil (8 vs. 28 weeks). Curing induced and increased crystallinity, and changes in the release rate, especially for the more crystalline matrix. In this case, both an increase and a decrease in release, were observed depending on the tempering condition. Curing at higher temperatures induced a melt-mediated crystallization and solid state transformation of the glycerol tristearate matrix and led to rearrangements of the inner structure with the formation of larger pores, which accelerated the release. In contrast, changes in the hydrogenated palm oil under the same curing conditions were less noticeable leading to a more robust formulation, because of less polymorphic changes over time. This study helps to understand the effect of lipid matrix composition and crystallinity degree on the performance of protein-loaded implants, and to establish criteria for the selection of a lipid carrier depending on the release profile desired. Copyright © 2018. Published by Elsevier B.V.

  2. In situ immobilization of proteins and RGD peptide on polyurethane surfaces via poly(ethylene oxide) coupling polymers for human endothelial cell growth.

    Science.gov (United States)

    Wang, Dong-an; Ji, Jian; Sun, Yong-hong; Shen, Jia-cong; Feng, Lin-xian; Elisseeff, Jennifer H

    2002-01-01

    A "CBABC"-type pentablock coupling polymer, mesylMPEO, was designed and synthesized to promote human endothelial cell growth on the surfaces of polyurethane biomaterials. The polymer was composed of a central 4,4'-methylenediphenyl diisocyanate (MDI) coupling unit and poly(ethylene oxide) (PEO) spacer arms with methanesulfonyl (mesyl) end groups pendent on both ends. As the presurface modifying additive (pre-SMA), the mesylMPEO was noncovalently introduced onto the poly(ether urethane) (PEU) surfaces by dip coating, upon which the protein/peptide factors (gelatin, albumin, and arginine-glycine-aspartic acid tripeptide [RGD]) were covalently immobilized in situ by cleavage of the original mesyl end groups. The pre-SMA synthesis and PEU surface modification were characterized using nuclear magnetic resonance spectroscopy ((1)H NMR), attenuated total reflection infrared spectroscopy (ATR-FTIR), and X-ray photoelectron spectroscopy (XPS). Human umbilical vein endothelial cells (HUVEC) were harvested manually by collagenase digestion and seeded on the modified PEU surfaces. Cell adhesion ratios (CAR) and cell proliferation ratios (CPR) were measured using flow cytometry, and the individual cell viability (ICV) was determined by MTT assay. The cell morphologies were investigated by optical inverted microscopy (OIM) and scanning electrical microscopy (SEM). The gelatin- and RGD-modified surfaces were HUVEC-compatible and promoted HUVEC growth. The albumin-modified surfaces were compatible but inhibited cell adhesion. The results also indicated that, for HUVEC in vitro cultivation, the cell adhesion stage was of particular importance and had a significant impact on the cell responses to the modified surfaces.

  3. Modified fractal iron oxide magnetic nanostructure: A novel and high performance platform for redox protein immobilization, direct electrochemistry and bioelectrocatalysis application.

    Science.gov (United States)

    Bagheri, Hasan; Ranjbari, Elias; Amiri-Aref, Mohaddeseh; Hajian, Ali; Ardakani, Yalda Hosseinzadeh; Amidi, Salimeh

    2016-11-15

    A novel biosensing platform based on fractal-pattern of iron oxides magnetic nanostructures (FIOMNs) and mixed hemi/ad-micelle of sodium dodecyl sulfate (SDS) was designed for the magnetic immobilization of hemoglobin (Hb) at a screen printed carbon electrode (SPCE). The FIOMNs was successfully synthesized through hydrothermal approach and characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM) and X-ray diffraction (XRD). In order to provide guidelines for the mixed hemi/ad-micelle formation, zeta-potential isotherms were investigated. The construction steps of the biosensor were evaluated by electrochemical impedance spectroscopy, cyclic voltammetry and Fourier transform infrared spectroscopy. Direct electron transfer of Hb incorporated into the biocomposite film was realized with a pair of quasi-reversible redox peak at the formal potential of -0.355V vs. Ag/AgCl attributing to heme Fe(III)/Fe(II) redox couple. The results suggested that synergistic functions regarding to the hyper-branched and multidirectional structure of FIOMNs and the dual interaction ability of mixed hemi/ad-micelle array of SDS molecules not only induce an effective electron transfer between the Hb and the underlying electrode (high heterogeneous electron transfer rate constant of 2.08s(-1)) but also provide powerful and special microenvironment for the adsorption of the redox proteins. Furthermore, the biosensor displayed an excellent performance to the electrocatalytic reduction of H2O2 with a detection limit of 0.48µM and Michaelis-Menten constant (Km) value of 44.2µM. The fabricated biosensor represented the features of sensitivity, disposable design, low sample volume, rapid and simple preparation step, and acceptable anti-interferences, which offer great perspectives for the screen-determination of H2O2 in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Platform for Lipid Based Nanocarriers' Formulation Components and their Potential Effects: A Literature Review.

    Science.gov (United States)

    Farid, Ragwa Mohamed; Youssef, Nancy Abdel Hamid Abou; Kassem, Abeer Ahmed

    2017-11-27

    Lipid based nanocarriers have gained recently enormous interest for pharmaceutical application. They have the potential to provide controlled drug release and to target the drug to a specific area. In addition, lipid based nanocarriers can improve the bioavailability of drugs suffering from high hepatic first-pass metabolism, by enhancing their transport via the lymphatic system. The main components of lipid based nanocarriers are lipids and surfactants. Both have great influence on the prepared lipid based systems characteristics. The criteria for their selection are much related to physicochemical properties of the drug and the required administration route. This work gives an overview on the effect of both the type and amount of lipids and surfactants used in the manufacture of lipid based nanocarriers on their behavior and characteristics. Recent studies revealed that the properties of the final product including; particle size, homogeneity, drug loading capacity, zeta potential, drug release profile, stability, permeability, pharmacokinetic properties, crystallinity and cytotoxicity, may be significantly influenced not only by the type but also the amount of the lipids and/or surfactants included in the formulation of the lipid based nanocarriers. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Immobilization of Peroxidase onto Magnetite Modified Polyaniline

    Directory of Open Access Journals (Sweden)

    Eduardo Fernandes Barbosa

    2012-01-01

    Full Text Available The present study describes the immobilization of horseradish peroxidase (HRP on magnetite-modified polyaniline (PANImG activated with glutaraldehyde. After the optimization of the methodology, the immobilization of HRP on PANImG produced the same yield (25% obtained for PANIG with an efficiency of 100% (active protein. The optimum pH for immobilization was displaced by the effect of the partition of protons produced in the microenvironment by the magnetite. The tests of repeated use have shown that PANImG-HRP can be used for 13 cycles with maintenance of 50% of the initial activity.

  6. In vitro digestibility of individual amino acids in rumen-undegraded protein: the modified three-step procedure and the immobilized digestive enzyme assay.

    Science.gov (United States)

    Boucher, S E; Calsamiglia, S; Parsons, C M; Stern, M D; Moreno, M Ruiz; Vázquez-Añón, M; Schwab, C G

    2009-08-01

    Three soybean meal, 3 SoyPlus (West Central Cooperative, Ralston, IA), 5 distillers dried grains with solubles, and 5 fish meal samples were used to evaluate the modified 3-step in vitro procedure (TSP) and the in vitro immobilized digestive enzyme assay (IDEA; Novus International Inc., St. Louis, MO) for estimating digestibility of AA in rumen-undegraded protein (RUP-AA). In a previous experiment, each sample was ruminally incubated in situ for 16 h, and in vivo digestibility of AA in the intact samples and in the rumen-undegraded residues (RUR) was obtained for all samples using the precision-fed cecectomized rooster assay. For the modified TSP, 5 g of RUR was weighed into polyester bags, which were then heat-sealed and placed into Daisy(II) incubator bottles. Samples were incubated in a pepsin/HCl solution followed by incubation in a pancreatin solution. After this incubation, residues remaining in the bags were analyzed for AA, and digestibility of RUP-AA was calculated based on disappearance from the bags. In vitro RUP-AA digestibility estimates obtained with this procedure were highly correlated to in vivo estimates. Corresponding intact feeds were also analyzed via the pepsin/pancreatin steps of the modified TSP. In vitro estimates of AA digestibility of the feeds were highly correlated to in vivo RUP-AA digestibility, which suggests that the feeds may not need to be ruminally incubated before determining RUP-AA digestibility in vitro. The RUR were also analyzed via the IDEA kits. The IDEA values of the RUR were good predictors of RUP-AA digestibility in soybean meal, SoyPlus, and distillers dried grains with solubles, but the IDEA values were not as good predictors of RUP-AA digestibility in fish meal. However, the IDEA values of intact feed samples were also determined and were highly correlated to in vivo RUP-AA digestibility for all feed types, suggesting that the IDEA value of intact feeds may be a better predictor of RUP-AA digestibility than the IDEA

  7. Metaproteomics Identifies the Protein Machinery Involved in Metal and Radionuclide Reduction in Subsurface Microbiomes and Elucidates Mechanisms and U(VI) Reduction Immobilization

    Energy Technology Data Exchange (ETDEWEB)

    Pfiffner, Susan M. [Univ. of Tennessee, Knoxville, TN (United States); Löffler, Frank [Univ. of Tennessee, Knoxville, TN (United States); Ritalahti, Kirsti [Univ. of Tennessee, Knoxville, TN (United States); Sayler, Gary [Univ. of Tennessee, Knoxville, TN (United States); Layton, Alice [Univ. of Tennessee, Knoxville, TN (United States); Hettich, Robert [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

    2015-08-31

    radionuclide reduction activity, are suitable biomarkers, (2) identify new biomarker targets for site assessment and bioremediation monitoring, and (3) provide new information about specific proteins and mechanisms involved in U(VI) reduction and immobilization. This expanded metagenomic and metaproteomic toolbox contributed to implementing science-driven site management with broad benefits to the DOE mission.

  8. Immobilized enzymes and cells

    Energy Technology Data Exchange (ETDEWEB)

    Bucke, C; Wiseman, A

    1981-04-04

    This article reviews the current state of the art of enzyme and cell immobilization and suggests advances which might be made during the 1980's. Current uses of immobilized enzymes include the use of glucoamylase in the production of glucose syrups from starch and glucose isomerase in the production of high fructose corn syrup. Possibilities for future uses of immobilized enzymes and cells include the utilization of whey and the production of ethanol.

  9. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes

    Science.gov (United States)

    Mohamad, Nur Royhaila; Marzuki, Nur Haziqah Che; Buang, Nor Aziah; Huyop, Fahrul; Wahab, Roswanira Abdul

    2015-01-01

    The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies. PMID:26019635

  10. Immobilized waste leaching

    International Nuclear Information System (INIS)

    Suarez, A.A.

    1989-01-01

    The main mechanism by which the immobilized radioactive materials can return to biosphere is the leaching due to the intrusion of water into the repositories. Some mathematical models and experiments utilized to evaluate the leaching rates in different immobilization matrices are described. (author) [pt

  11. Haloalkane hydrolysis with an immobilized haloalkane dehalogenase.

    Science.gov (United States)

    Dravis, B C; Swanson, P E; Russell, A J

    2001-11-20

    Haloalkane dehalogenase from Rhodococcus rhodochrous was covalently immobilized onto a polyethyleneimine impregnated gamma-alumina support. The dehalogenating enzyme was found to retain greater than 40% of its original activity after immobilization, displaying an optimal loading (max. activity/supported protein) of 70 to 75 mg/g with an apparent maximum (max. protein/support) of 156 mg/g. The substrate, 1,2,3-trichloropropane, was found to favorably partition (adsorb) onto the inorganic alumina carrier (10 to 20 mg/g), thereby increasing the local reactant concentration with respect to the catalyst's environment, whereas the product, 2,3-dichloropropan-1-ol, demonstrated no affinity. Additionally, the inorganic alumina support exhibited no adverse effects because of solvent/component incompatibilities or deterioration due to pH variance (pH 7.0 to 10.5). As a result of the large surface area to volume ratio of the support matrix and the accessibility of the bound protein, the immobilized biocatalyst was not subject to internal mass transfer limitations. External diffusional restrictions could be eliminated with simple agitation (mixing speed: 50 rpm; flux: 4.22 cm/min). The pH-dependence of the immobilized dehalogenase was essentially the same as that for the native enzyme. Finally, both the thermostability and resistance toward inactivation by organic solvent were improved by more than an order of magnitude after immobilization. Copyright 2001 John Wiley & Sons, Inc.

  12. Toward the establishment of standardized in vitro tests for lipid-based formulations, part 3

    DEFF Research Database (Denmark)

    Williams, Hywel D; Sassene, Philip; Kleberg, Karen

    2013-01-01

    PURPOSE: Recent studies have shown that digestion of lipid-based formulations (LBFs) can stimulate both supersaturation and precipitation. The current study has evaluated the drug, formulation and dose-dependence of the supersaturation - precipitation balance for a range of LBFs. METHODS: Type I,...

  13. Lipid-based liquid biofuels from autotrophic microalgae: energetic and environmental performance

    NARCIS (Netherlands)

    Reijnders, L.

    2013-01-01

    Commercial cultivation of autotrophic microalgae for food production dates back to the 1950s. Autotrophic microalgae have also been proposed as a source for lipid-based liquid biofuels. As yet, there is no commercial production of such biofuels and estimated near-term prices are far in excess of

  14. Plutonium Disposition by Immobilization

    International Nuclear Information System (INIS)

    Gould, T.; DiSabatino, A.; Mitchell, M.

    2000-01-01

    The ultimate goal of the Department of Energy (DOE) Immobilization Project is to develop, construct, and operate facilities that will immobilize between 17 to 50 tonnes (MT) of U.S. surplus weapons-usable plutonium materials in waste forms that meet the ''spent fuel'' standard and are acceptable for disposal in a geologic repository. Using the ceramic can-in-canister technology selected for immobilization, surplus plutonium materials will be chemically combined into ceramic forms which will be encapsulated within large canisters of high level waste (HLW) glass. Deployment of the immobilization capability should occur by 2008 and be completed within 10 years. In support of this goal, the DOE Office of Fissile Materials Disposition (MD) is conducting development and testing (D and T) activities at four DOE laboratories under the technical leadership of Lawrence Livermore National Laboratory (LLNL). The Savannah River Site has been selected as the site for the planned Plutonium Immobilization Plant (PIP). The D and T effort, now in its third year, will establish the technical bases for the design, construction, and operation of the U. S. capability to immobilize surplus plutonium in a suitable and cost-effective manner. Based on the D and T effort and on the development of a conceptual design of the PIP, automation is expected to play a key role in the design and operation of the Immobilization Plant. Automation and remote handling are needed to achieve required dose reduction and to enhance operational efficiency

  15. Chemically modified, immobilized trypsin reactor with improved digestion efficiency

    NARCIS (Netherlands)

    Freije, J.R.; Mulder, P.P.; Werkman, W.; Rieux, L.; Niederlander, H.A G; Verpoorte, Sabeth; Bischoff, Rainer

    2005-01-01

    Tryptic digestion followed by identification using mass spectrometry is an important step in many proteomic studies. Here, we describe the preparation of immobilized, acetylated trypsin for enhanced digestion efficacy in integrated protein analysis platforms. Complete digestion of cytochrome c was

  16. Current and emerging lipid-based systems for transdermal drug delivery.

    Science.gov (United States)

    Singla, Sumeet K; Sachdeva, Vishal

    2015-01-01

    Developing a transdermal drug delivery system is a challenging task considering the selective permeability of the skin and the physicochemical properties the drug must possess to permeate through the skin. Lipid-based drug delivery systems have contributed a great deal in this direction in the last few decades, and thereby have helped to expand the range of therapeutic molecules that can be delivered through the skin in a safe and effective manner. Additionally, vesicular delivery systems such as nanoparticles and emulsions have also played important roles in providing alternative novel approaches for drug delivery. In this article, we will discuss some of the current and future lipid-based systems for transdermal drug delivery along with the associated challenges.

  17. Tendon collagen synthesis declines with immobilization in elderly humans

    DEFF Research Database (Denmark)

    Dideriksen, Kasper; Boesen, Anders P; Reitelseder, Søren

    2017-01-01

    -80 yr) were randomly assigned to NSAIDs (ibuprofen 1,200 mg/day; Ibu) or placebo (Plc). One lower limb was immobilized in a cast for 2 wk and retrained for 6 wk. Tendon collagen protein synthesis, mechanical properties, size, expression of genes related to collagen turnover and remodeling, and signal...... intensity (from magnetic resonance imaging) were investigated. Tendon collagen synthesis decreased (P ... immobilization in both groups, whereas scleraxis mRNA decreased with inactivity in the Plc group only (P collagen protein synthesis decreased after 2 wk of immobilization, whereas tendon stiffness and modulus were only marginally reduced, and NSAIDs had no influence upon this...

  18. Reversible thermal denaturation of immobilized rhodanese

    International Nuclear Information System (INIS)

    Horowitz, P.; Bowman, S.

    1987-01-01

    For the first time, the enzyme rhodanese had been refolded after thermal denaturation. This was previously not possible because of the strong tendency for the soluble enzyme to aggregate at temperatures above 37 degrees C. The present work used rhodanese that was covalently coupled to a solid support under conditions that were found to preserve enzyme activity. Rhodanese was immobilized using an N-hydroxymalonimidyl derivative of Sepharose containing a 6-carbon spacer. The number of immobilized competent active sites was measured by using [ 35 S]SO 3 (2-) to form an active site persulfide that is the obligatory catalytic intermediate. Soluble enzyme was irreversibly inactivated in 10 min at 52 degrees C. The immobilized enzyme regained at least 30% of its original activity even after boiling for 20 min. The immobilized enzyme had a Km and Vmax that were each approximately 3 times higher than the corresponding values for the native enzyme. After preincubation at high temperatures, progress curves for the immobilized enzyme showed induction periods of up to 5 min before attaining apparently linear steady states. The pH dependence of the activity was the same for both the soluble and the immobilized enzyme. These results indicate significant stabilization of rhodanese after immobilization, and instabilities caused by adventitious solution components are not the sole reasons for irreversibility of thermal denaturation seen with the soluble enzyme. The results are consistent with models for rhodanese that invoke protein association as a major cause of inactivation of the enzyme. Furthermore, the induction period in the progress curves is consistent with studies which show that rhodanese refolding proceeds through intermediate states

  19. A simple and robust approach to immobilization of antibody fragments.

    Science.gov (United States)

    Ikonomova, Svetlana P; He, Ziming; Karlsson, Amy J

    2016-08-01

    Antibody fragments, such as the single-chain variable fragment (scFv), have much potential in research and diagnostics because of their antigen-binding ability similar to a full-sized antibody and their ease of production in microorganisms. Some applications of antibody fragments require immobilization on a surface, and we have established a simple immobilization method that is based on the biotin-streptavidin interaction and does not require a separate purification step. We genetically fused two biotinylation tags-the biotin carboxyl carrier protein (BCCP) or the AviTag minimal sequence-to six different scFvs (scFv13R4, scFvD10, scFv26-10, scFv3, scFv5, and scFv12) for site-specific biotinylation in vivo by endogenous biotin ligases produced by Escherichia coli. The biotinylated scFvs were immobilized onto streptavidin-coated plates directly from cell lysates, and immobilization was detected through enzyme-linked immunosorbent assays. All scFvs fusions were successfully immobilized, and scFvs biotinylated via the BCCP tag tended to immobilize better than those biotinylated via the AviTag, even when biotinylation efficiency was improved with the biotin ligase BirA. The ability of immobilized scFvs to bind antigens was confirmed using scFv13R4 and scFvD10 with their respective targets β-galactosidase and bacteriophage lambda head protein D (gpD). The immobilized scFv13R4 bound to β-galactosidase at the same level for both biotinylation tags when the surface was saturated with the scFv, and immobilized scFvs retained their functionality for at least 100days after immobilization. The simplicity and robustness of our method make it a promising approach for future applications that require antibody fragment immobilization. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Oriented immobilization of His-tagged kinase RIO1 protein on redox active N-(IDA-like)-Cu(II) monolayer deposited on gold electrode—The base of electrochemical biosensor

    International Nuclear Information System (INIS)

    Mielecki, Marcin; Wojtasik, Justyn; Zborowska, Magdalena; Kurzątkowska, Katarzyna; Grzelak, Krystyna; Dehaen, Wim; Radecki, Jerzy; Radecka, Hanna

    2013-01-01

    Highlights: ► The redox active N-(IDA-like)-Cu(II) monolayer is suitable for oriented and stable immobilization of His-tagged kinase Rio1. ► Cu(II) deposited onto the electrode surface play double role: immobilization sites for His-tagged proteins and transduction centres tracking the protein–small molecule interactions. ► The base of biosensor response towards target compound is the change of Rio1 conformation lading to alternation of the permeability of counter ions to Cu(II) redox centres. -- Abstract: The fabrication of electrochemical biosensor consists of the following successive steps: formation of thiol derivative of iminodiacetic acid (IDA-like/N-heterocyclic donor) and N-acetylcysteamine (NAC) self-assembled monolayer on the Au electrode, complexation of Cu(II) by N(IDA-like) attached to the surface of the Au electrode and immobilization of kinase protein Rio1 through N(IDA-like)-Cu(II)-histidine-tag covalent bond formation. Each step of modification was controlled by cyclic voltammetry, electrochemical impedance spectrometry and atomic force microscopy. The interactions between rHis 6 -Rio1 attached to the surface of the electrode and tyrphostin inhibitor (2E)-N-Benzyl-2-cyano-3-(3,4-dihydroxyphenyl)-acrylamide (AG-490) and its analogue (2-cyano-N-(4-methoxyphenyl)-3-(pyridin-3-yl)prop-2-enamide) (CPE), present in aqueous solution were monitored with Osteryoung square wave voltammetry. The basis of the biosensor response was the change in the electrochemical properties of Cu(II) redox centres upon formation of the rHis 6 -Rio1-inhibitor complex. A linear responses with high reproducibility and stability were observed between 0.10 and 0.40 μM of AG-490 as well as of CPE. The interaction between rHis 6 -Rio1 and AG-490 was stronger than the interaction with its analogue CPE. Cu(II) redox current decrease of 37.9 ± 1.6% and 23.3 ± 1.0% were observed in the presence of 0.40 μM of AG-490 and CPE, respectively. The presented biosensor could be

  1. Effect of photo-immobilization of epidermal growth factor on the cellular behaviors

    International Nuclear Information System (INIS)

    Ogiwara, Kazutaka; Nagaoka, Masato; Cho, Chong-Su; Akaike, Toshihiro

    2006-01-01

    We constructed photo-reactive epidermal growth factor (EGF) bearing p-azido phenylalanine at the C-terminal (HEGFP) by genetic engineering to investigate the possibility of immobilized EGF as a novel artificial extracellular matrix (ECM). The constructed recombinant protein was immobilized to glass surface by ultraviolet irradiation. A431 cells adhered both to HEGFP-immobilized and collagen-coated surfaces. Interaction between immobilized HEGFP and EGF receptors in the A431 cells was independent of Mg 2+ although integrin-mediated cell adhesion to natural ECMs is dependent on Mg 2+ . Phosphorylation of EGF receptors in A431 cells was induced by immobilized HEGFP as same as soluble EGF. DNA uptake of hepatocytes decreased by immobilized HEGFP whereas it increased by soluble EGF. Liver-specific functions of hepatocytes were maintained for 3 days by immobilized HEGFP whereas they were not maintained by soluble EGF, indicating that immobilized HEGFP follows different signal transduction pathway from soluble EGF

  2. Immobilization of microorganisms. Part 1. Preparation of immobilized Lactobacillus bulgaricus

    Energy Technology Data Exchange (ETDEWEB)

    Lee, K H

    1981-01-01

    The immobilization of Lactobacillus bulgaricus on polyacrylamide and on alginate beads was investigated. The most active immobilized cells were obtained by entrapment in Ca alginate beads. These immobilized microbial cells, when introduced into 4.5% lactose solution and whey solution showed maximum relative activity of 28% for lactose and 18% for whey compared to free cells.

  3. Halloysite Clay Nanotubes for Enzyme Immobilization.

    Science.gov (United States)

    Tully, Joshua; Yendluri, Raghuvara; Lvov, Yuri

    2016-02-08

    Halloysite clay is an aluminosilicate nanotube formed by rolling flat sheets of kaolinite clay. They have a 15 nm lumen, 50-70 nm external diameter, length of 0.5-1 μm, and different inside/outside chemistry. Due to these nanoscale properties, they are used for loading, storage, and controlled release of active chemical agents, including anticorrosions, biocides, and drugs. We studied the immobilization in halloysite of laccase, glucose oxidase, and lipase. Overall, negatively charged proteins taken above their isoelectric points were mostly loaded into the positively charged tube's lumen. Typical tube loading with proteins was 6-7 wt % from which one-third was released in 5-10 h and the other two-thirds remained, providing enhanced biocatalysis in nanoconfined conditions. Immobilized lipase showed enhanced stability at acidic pH, and the optimum pH shifted to more alkaline pH. Immobilized laccase was more stable with respect to time, and immobilized glucose oxidase showed retention of enzymatic activity up to 70 °C, whereas the native sample was inactive.

  4. Protein and polymer immobilized La{sub 0.7}Sr{sub 0.3}MnO{sub 3} nanoparticles for possible biomedical applications

    Energy Technology Data Exchange (ETDEWEB)

    Bhayani, K R [Agharkar Research Institute, G G Agarkar Road, Pune 411004 (India); Kale, S N [Agharkar Research Institute, G G Agarkar Road, Pune 411004 (India); Arora, Sumit [Agharkar Research Institute, G G Agarkar Road, Pune 411004 (India); Rajagopal, Rajashree [Computer Science Unit, Fergusson College, Pune 411004 (India); Mamgain, H [Agharkar Research Institute, G G Agarkar Road, Pune 411004 (India); Kaul-Ghanekar, R [Agharkar Research Institute, G G Agarkar Road, Pune 411004 (India); Kundaliya, Darshan C [Centre for Superconductivity Research, University of Maryland, College Park, MD 20742 (United States); Kulkarni, S D [Centre for Materials Characterization, National Chemical Laboratory, Pune 411008 (India); Pasricha, Renu [Centre for Materials Characterization, National Chemical Laboratory, Pune 411008 (India); Dhole, S D [Department of Physics, University of Pune, Pune 411007 (India); Ogale, S B [Physical and Materials Chemistry Division, National Chemical Laboratory, Pune 411008 (India); Paknikar, K M [Agharkar Research Institute, G G Agarkar Road, Pune 411004 (India)

    2007-08-29

    La{sub 0.7}Sr{sub 0.3}MnO{sub 3} (LSMO) is a mixed-valent room temperature ferromagnet with properties that are attractive for their applicability in biomedicine. We report, for the first time, immobilization of commonly used biocompatible molecules on LSMO nanoparticles, namely bovine serum albumin and dextran. The former was conjugated to LSMO using 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (CDI) as a coupling agent while the latter was used without any coupler. These bioconjugated nanoparticles exhibit several properties that suggest their applicability in the field of biomedicine, namely (a) no changes in the Curie temperature at {approx}360 K after conjugation with biomolecules, (b) rapid attainment of the desired temperature (48 deg. C) at low concentration (e.g. fluidized dextran-coated system at 80 {mu}g ml{sup -1}) upon exposure to 20 MHz radio-frequency, (c) extremely low cytotoxicity in skin carcinoma, human fibrosarcoma and neuroblastoma cell lines and (d) high stability of the LSMO system with negligible leaching of ionic manganese into the delivery medium, indicating their safety in possible human applications.

  5. Limb immobilization and corticobasal syndrome.

    Science.gov (United States)

    Graff-Radford, Jonathan; Boeve, Bradley F; Drubach, Daniel A; Knopman, David S; Ahlskog, J Eric; Golden, Erin C; Drubach, Dina I; Petersen, Ronald C; Josephs, Keith A

    2012-12-01

    Recently, we evaluated two patients with corticobasal syndrome (CBS) who reported symptom onset after limb immobilization. Our objective was to investigate the association between trauma, immobilization and CBS. The charts of forty-four consecutive CBS patients seen in the Mayo Clinic Alzheimer Disease Research Center were reviewed with attention to trauma and limb immobilization. 10 CBS patients (23%) had immobilization or trauma on the most affected limb preceding the onset or acceleration of symptoms. The median age at onset was 61. Six patients manifested their first symptoms after immobilization from surgery or fracture with one after leg trauma. Four patients had pre-existing symptoms of limb dysfunction but significantly worsened after immobilization or surgery. 23 percent of patients had immobilization or trauma of the affected limb. This might have implications for management of CBS, for avoiding injury, limiting immobilization and increasing movement in the affected limb. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Lipid-based formulations for oral administration of poorly water-soluble drugs

    DEFF Research Database (Denmark)

    Mu, Huiling; Holm, René; Müllertz, Anette

    2013-01-01

    Lipid-based drug delivery systems have shown great potentials in oral delivery of poorly water-soluble drugs, primarily for lipophilic drugs, with several successfully marketed products. Pre-dissolving drugs in lipids, surfactants, or mixtures of lipids and surfactants omits the dissolving....../dissolution step, which is a potential rate limiting factor for oral absorption of poorly water-soluble drugs. Lipids not only vary in structures and physiochemical properties, but also in their digestibility and absorption pathway; therefore selection of lipid excipients and dosage form has a pronounced effect...

  7. Clinical studies with oral lipid based formulations of poorly soluble compounds

    DEFF Research Database (Denmark)

    Fatouros, Dimitrios; Karpf, Ditte M; Nielsen, Flemming S

    2007-01-01

    . Several drug products intended for oral administration have been marketed utilizing lipid and surfactant based formulations. Sandimmune((R)) and Sandimmune Neoral((R)) (cyclosporin A, Novartis), Norvir((R)) (ritonavir), and Fortovase((R)) (saquinavir) have been formulated in self-emulsifying drug delivery...... systems (SEDDS). This review summarizes published pharmacokinetic studies of orally administered lipid based formulations of poorly aqueous soluble drugs in human subjects. Special attention has been paid to the physicochemical characteristics of the formulations, when available and the impact...

  8. Lipid-Based Nanovectors for Targeting of CD44-Overexpressing Tumor Cells

    Directory of Open Access Journals (Sweden)

    Silvia Arpicco

    2013-01-01

    Full Text Available Hyaluronic acid (HA is a naturally occurring glycosaminoglycan that exists in living systems, and it is a major component of the extracellular matrix. The hyaluronic acid receptor CD44 is found at low levels on the surface of epithelial, haematopoietic, and neuronal cells and is overexpressed in many cancer cells particularly in tumour initiating cells. HA has been therefore used as ligand attached to HA-lipid-based nanovectors for the active targeting of small or large active molecules for the treatment of cancer. This paper describes the different approaches employed for the preparation, characterization, and evaluation of these potent delivery systems.

  9. Calibrating passive sampling and passive dosing techniques to lipid based concentrations

    DEFF Research Database (Denmark)

    Mayer, Philipp; Schmidt, Stine Nørgaard; Annika, A.

    2011-01-01

    Equilibrium sampling into various formats of the silicone polydimethylsiloxane (PDMS) is increasingly used to measure the exposure of hydrophobic organic chemicals in environmental matrices, and passive dosing from silicone is increasingly used to control and maintain their exposure in laboratory...... coated vials and with Head Space Solid Phase Microextraction (HS-SPME) yielded lipid based concentrations that were in good agreement with each other, but about a factor of two higher than measured lipid-normalized concentrations in the organisms. Passive dosing was applied to bioconcentration...

  10. Membranes suited for immobilizing biomolecules

    NARCIS (Netherlands)

    2009-01-01

    The present invention relates to flow-through membranes suitable for the immobilization of biomols., methods for the prepn. of such membranes and the use of such membranes for the immobilization of biomols. and subsequent detection of immobilized biomols. The invention concerns a flow-through

  11. Compound immobilization and drug-affinity chromatography.

    Science.gov (United States)

    Rix, Uwe; Gridling, Manuela; Superti-Furga, Giulio

    2012-01-01

    Bioactive small molecules act through modulating a yet unpredictable number of targets. It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to understanding its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identification of a target protein will be detrimental to binding affinity. Also, many molecules are sensitive to heat or to certain chemicals, such as acid or base, and might be destroyed during the process of immobilization, which therefore needs to be not only efficient, but also mild. The subsequent affinity chromatography step needs to preserve molecular and conformational integrity of both bait compound and proteins in order to result in the desired specific enrichment while ensuring a high level of compatibility with downstream analysis by mass spectrometry. Thus, the right choice of detergent, buffer, and protease inhibitors is also essential. This chapter describes a widely applicable procedure for the immobilization of small molecule drugs and for drug-affinity chromatography with subsequent protein identification by mass spectrometry.

  12. Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency

    Directory of Open Access Journals (Sweden)

    Helena Sork

    2016-01-01

    Full Text Available The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.

  13. Development of New Lipid-Based Paclitaxel Nanoparticles Using Sequential Simplex Optimization

    Science.gov (United States)

    Dong, Xiaowei; Mattingly, Cynthia A.; Tseng, Michael; Cho, Moo; Adams, Val R.; Mumper, Russell J.

    2008-01-01

    The objective of these studies was to develop Cremophor-free lipid-based paclitaxel (PX) nanoparticle formulations prepared from warm microemulsion precursors. To identify and optimize new nanoparticles, experimental design was performed combining Taguchi array and sequential simplex optimization. The combination of Taguchi array and sequential simplex optimization efficiently directed the design of paclitaxel nanoparticles. Two optimized paclitaxel nanoparticles (NPs) were obtained: G78 NPs composed of glyceryl tridodecanoate (GT) and polyoxyethylene 20-stearyl ether (Brij 78), and BTM NPs composed of Miglyol 812, Brij 78 and D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS). Both nanoparticles successfully entrapped paclitaxel at a final concentration of 150 μg/ml (over 6% drug loading) with particle sizes less than 200 nm and over 85% of entrapment efficiency. These novel paclitaxel nanoparticles were stable at 4°C over three months and in PBS at 37°C over 102 hours as measured by physical stability. Release of paclitaxel was slow and sustained without initial burst release. Cytotoxicity studies in MDA-MB-231 cancer cells showed that both nanoparticles have similar anticancer activities compared to Taxol®. Interestingly, PX BTM nanocapsules could be lyophilized without cryoprotectants. The lyophilized powder comprised only of PX BTM NPs in water could be rapidly rehydrated with complete retention of original physicochemical properties, in-vitro release properties, and cytotoxicity profile. Sequential Simplex Optimization has been utilized to identify promising new lipid-based paclitaxel nanoparticles having useful attributes. PMID:19111929

  14. Lipid-Based Liquid Crystalline Nanoparticles Facilitate Cytosolic Delivery of siRNA via Structural Transformation.

    Science.gov (United States)

    He, Shufang; Fan, Weiwei; Wu, Na; Zhu, Jingjing; Miao, Yunqiu; Miao, Xiaran; Li, Feifei; Zhang, Xinxin; Gan, Yong

    2018-04-11

    RNA interference (RNAi) technology has shown great promise for the treatment of cancer and other genetic disorders. Despite the efforts to increase the target tissue distribution, the safe and effective delivery of siRNA to the diseased cells with sufficient cytosolic transport is another critical factor for successful RNAi clinical application. Here, the constructed lipid-based liquid crystalline nanoparticles, called nano-Transformers, can transform thestructure in the intracellular acidic environment and perform high-efficient siRNA delivery for cancer treatment. The developed nano-Transformers have satisfactory siRNA loading efficiency and low cytotoxicity. Different from the traditional cationic nanocarriers, the endosomal membrane fusion induced by the conformational transition of lipids contributes to the easy dissociation of siRNA from nanocarriers and direct release of free siRNA into cytoplasm. We show that transfection with cyclin-dependent kinase 1 (CDK1)-siRNA-loaded nano-Transformers causes up to 95% reduction of relevant mRNA in vitro and greatly inhibits the tumor growth without causing any immunogenic response in vivo. This work highlights that the lipid-based nano-Transformers may become the next generation of siRNA delivery system with higher efficacy and improved safety profiles.

  15. (Au/PANA/PVAc) nanofibers as a novel composite matrix for albumin and streptavidin immobilization

    Energy Technology Data Exchange (ETDEWEB)

    Golshaei, Rana [University of Kashan, Institute of Nano Science and Nano Technology, Kashan, P.O. Box 87317-51167, Islamic Republic of Iran (Iran, Islamic Republic of); Guler, Zeliha [Istanbul Technical University, Nanoscience and Nanoengineering, Maslak, Istanbul 34469 (Turkey); Sarac, Sezai A., E-mail: sarac@itu.edu.tr [Istanbul Technical University, Nanoscience and Nanoengineering, Maslak, Istanbul 34469 (Turkey); Istanbul Technical University, Department of Chemistry and Polymer Science and Technology, Maslak, Istanbul 34469 (Turkey)

    2016-03-01

    A novel electrospun nanofiber mat (Au/PANA/PVAc) consists of (Gold/Poly Anthranilic acid) (Au/PANA) core/shell nanostructures as a support material for protein immobilization that was developed and characterized by electrochemical impedance spectroscopy. In the core/shells, PANA served carboxyl groups (− COOH) for covalent protein immobilization and Au enhanced the electrochemical properties by acting as tiny conduction centers to facilitate electron transfer. Covalent immobilization of albumin and streptavidin as model proteins onto the (Au/PANA/PVAc) nanofibers was carried out by using 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxyl succinimide (NHS) activation. PVAc nanofibers were compared with Au/PANA/PVAc nanofibers before and after protein immobilization. The successful covalent binding of both albumin and streptavidin onto (Au/PANA/PVAc) nanofibers was confirmed by FTIR-ATR, Electron Microscopy/Energy-Dispersive X-ray Spectroscopy SEM/EDX and Electrochemical impedance spectroscopy (EIS). The nanofibers became resistive due to protein immobilization and the higher charge transfer resistance was observed after higher amount of protein was immobilized. - Highlights: • Au/PANA/PVAc nanofibers with (COOH) groups as a suitable supports for covalent immobilization of proteins. • Increasing of the resistivity of the nanofibers after immobilization of the proteins. • Activation of Au/PANA/PVAc nanofibers by using EDC/NHS.

  16. Immobilization of bioactive fibroblast growth factor-2 into cubic proteinous microcrystals (Bombyx mori cypovirus polyhedra) that are insoluble in a physiological cellular environment.

    Science.gov (United States)

    Mori, Hajime; Shukunami, Chisa; Furuyama, Akiko; Notsu, Hiroyuki; Nishizaki, Yuriko; Hiraki, Yuji

    2007-06-08

    The supramolecular architecture of the extracellular matrix and the disposition of its specific accessory molecules give rise to variable heterotopic signaling cues for single cells. Here we have described the successful occlusion of human fibroblast growth factor-2 (FGF-2) into the cubic inclusion bodies (FGF-2 polyhedra) of the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). The polyhedra are proteinous cubic crystals of several microns in size that are insoluble in the extracellular milieu. Purified FGF-2 polyhedra were found to stimulate proliferation and phosphorylation of p44/p42 mitogen-activated protein kinase in cultured fibroblasts. Moreover, cellular responses were blocked by a synthetic inhibitor of the FGF signaling pathway, SU5402, suggesting that FGF-2 polyhedra indeed act through FGF receptors. Furthermore, FGF-2 polyhedra retained potent growth stimulatory properties even after desiccation. We have demonstrated that BmCPV polyhedra microcrystals that occlude extracellular signaling proteins are a novel and versatile tool that can be employed to analyze cellular behavior at the single cell level.

  17. Immobilization of xanthine oxidase on a polyaniline silicone support.

    Science.gov (United States)

    Nadruz, W; Marques, E T; Azevedo, W M; Lima-Filho, J L; Carvalho, L B

    1996-03-01

    A polyaniline silicone support to immobilize xanthine oxidase is proposed as a reactor coil to monitor the action of xanthine oxidase on hypoxanthine, xanthine and 6-mercaptopurine. A purified xanthine oxidase immobilized on this support lost 80% of the initial activity after 12 min of use. Co-immobilization of superoxide dismutase and catalase increased the stability of immobilized xanthine oxidase so that the derivative maintained 79% of its initial activity after 4.6 h of continuous use in which 1.5 mumol purine bases were converted by the immobilized enzyme system. There is no evidence of either polyaniline or protein leaching from the coil during 3 h of continuous use. When solutions (10 ml) of hypoxanthine, xanthine and 6-mercaptopurine were circulated individually through the xanthine oxidase-superoxide dismutase-catalase-polyaniline coil (1 mm internal diameter and 3 m in length, 3 ml internal volume) activities of 8.12, 11.17 and 1.09 nmol min-1 coil-1, respectively, were obtained. The advantages of the reactor configuration and the redox properties of the polymer, particularly with respect to immobilized oxidoreductases, make this methodology attractive for similar enzyme systems. This immobilized enzyme system using polyaniline-silicone as support converted 6-mercaptopurine to 6-thiouric acid with equal efficiency as resins based on polyacrylamide and polyamide 11.

  18. Toward the establishment of standardized in vitro tests for lipid-based formulations, part 4

    DEFF Research Database (Denmark)

    Williams, Hywel D; Sassene, Philip; Kleberg, Karen

    2014-01-01

    The Lipid Formulation Classification System Consortium looks to develop standardized in vitro tests and to generate much-needed performance criteria for lipid-based formulations (LBFs). This article highlights the value of performing a second, more stressful digestion test to identify LBFs near...... a performance threshold and to facilitate lead formulation selection in instances where several LBF prototypes perform adequately under standard digestion conditions (but where further discrimination is necessary). Stressed digestion tests can be designed based on an understanding of the factors that affect LBF...... development, and facilitate dialogue with the regulatory authorities. This classification system is based on the concept that performance evaluations across three in vitro tests, designed to subject a LBF to progressively more challenging conditions, will enable effective LBF discrimination and performance...

  19. Lipidomics in translational research and the clinical significance of lipid-based biomarkers.

    Science.gov (United States)

    Stephenson, Daniel J; Hoeferlin, L Alexis; Chalfant, Charles E

    2017-11-01

    Lipidomics is a rapidly developing field of study that focuses on the identification and quantitation of various lipid species in the lipidome. Lipidomics has now emerged in the forefront of scientific research due to the importance of lipids in metabolism, cancer, and disease. Using both targeted and untargeted mass spectrometry as a tool for analysis, progress in the field has rapidly progressed in the last decade. Having the ability to assess these small molecules in vivo has led to better understanding of several lipid-driven mechanisms and the identification of lipid-based biomarkers in neurodegenerative disease, cancer, sepsis, wound healing, and pre-eclampsia. Biomarker identification and mechanistic understanding of specific lipid pathways linked to a disease's pathologies can form the foundation in the development of novel therapeutics in hopes of curing human disease. Published by Elsevier Inc.

  20. Implications of formulation design on lipid-based nanostructured carrier system for drug delivery to brain.

    Science.gov (United States)

    Salunkhe, Sachin S; Bhatia, Neela M; Bhatia, Manish S

    2016-05-01

    The aim of present investigation was to formulate and develop lipid-based nanostructured carriers (NLCs) containing Idebenone (IDE) for delivery to brain. Attempts have been made to evaluate IDE NLCs for its pharmacokinetic and pharmacodynamic profile through the objective of enhancement in bioavailability and effectivity of drug. Nanoprecipitation technique was used for development of drug loaded NLCs. The components solid lipid Precirol ATO 5, oil Miglyol 840, surfactants Tween 80 and Labrasol have been screened out for formulation development by consideration of preformulation parameters including solubility, Required Hydrophilic lipophilic balance (HLB) of lipids and stability study. Developed IDE NLCs were subjected for particle size, zeta potential, entrapment efficiency (%EE), crystallographic investigation, transmission electron microscopy, in vitro drug release, pharmacokinetics, in vivo and stability study. Formulation under investigation has particle size 174.1 ± 2.6 nm, zeta potential -18.65 ± 1.13 mV and% EE 90.68 ± 2.90. Crystallographic studies exemplified for partial amorphization of IDE by molecularly dispersion within lipid crust. IDE NLCs showed drug release 93.56 ± 0.39% at end of 24 h by following Higuchi model which necessitates for appropriate drug delivery with enhancement in bioavailability of drug by 4.6-fold in plasma and 2.8-fold in brain over plain drug loaded aqueous dispersions. In vivo studies revealed that effect of drug was enhanced by prepared lipid nanocarriers. IDE lipid-based nanostructured carriers could have potential for efficient drug delivery to brain with enhancement in bioavailability of drug over the conventional formulations.

  1. Prenatal lipid-based nutrient supplements increase cord leptin concentration in pregnant women from rural Burkina Faso.

    Science.gov (United States)

    Huybregts, Lieven; Roberfroid, Dominique; Lanou, Hermann; Meda, Nicolas; Taes, Youri; Valea, Innocent; D'Alessandro, Umberto; Kolsteren, Patrick; Van Camp, John

    2013-05-01

    In developing countries, prenatal lipid-based nutrient supplements (LNSs) were shown to increase birth size; however, the mechanism of this effect remains unknown. Cord blood hormone concentrations are strongly associated with birth size. Therefore, we hypothesize that LNSs increase birth size through a change in the endocrine regulation of fetal development. We compared the effect of daily prenatal LNSs with multiple micronutrient tablets on cord blood hormone concentrations using a randomized, controlled design including 197 pregnant women from rural Burkina Faso. Insulin-like growth factors (IGF) I and II, their binding proteins IGFBP-1 and IGFBP-3, leptin, cortisol, and insulin were quantified in cord sera using immunoassays. LNS was associated with higher cord blood leptin mainly in primigravidae (+57%; P = 0.02) and women from the highest tertile of BMI at study inclusion (+41%; P = 0.02). We did not find any significant LNS effects on other measured cord hormones. The observed increase in cord leptin was associated with a significantly higher birth weight. Cord sera from small-for-gestational age newborns had lower median IGF-I (-9 μg/L; P = 0.003), IGF-II (-79 μg/L; P = 0.003), IGFBP-3 (-0.7 μg/L; P = 0.007), and leptin (-1.0 μg/L; P = 0.016) concentrations but higher median cortisol (+18 μg/L; P = 0.037) concentrations compared with normally grown newborns. Prenatal LNS resulted in increased cord leptin concentrations in primigravidae and mothers with higher BMI at study inclusion. The elevated leptin concentrations could point toward a higher neonatal fat mass.

  2. Immediate-early gene response to repeated immobilization: Fos protein and arc mRNA levels appear to be less sensitive than c-fos mRNA to adaptation.

    Science.gov (United States)

    Ons, Sheila; Rotllant, David; Marín-Blasco, Ignacio J; Armario, Antonio

    2010-06-01

    Stress exposure resulted in brain induction of immediate-early genes (IEGs), considered as markers of neuronal activation. Upon repeated exposure to the same stressor, reduction of IEG response (adaptation) has been often observed, but there are important discrepancies in literature that may be in part related to the particular IEG and methodology used. We studied the differential pattern of adaptation of the IEGs c-fos and arc (activity-regulated cytoskeleton-associated protein) after repeated exposure to a severe stressor: immobilization on wooden boards (IMO). Rats repeatedly exposed to IMO showed reduced c-fos mRNA levels in response to acute IMO in most brain areas studied: the medial prefrontal cortex (mPFC), lateral septum (LS), medial amygdala (MeA), paraventricular nucleus of the hypothalamus (PVN) and locus coeruleus. In contrast, the number of neurons showing Fos-like immunoreactivity was only reduced in the MeA and the various subregions of the PVN. IMO-induced increases in arc gene expression were restricted to telencephalic regions and reduced by repeated IMO only in the mPFC. Double-labelling in the LS of IMO-exposed rats revealed that arc was expressed in only one-third of Fos+ neurons, suggesting two populations of Fos+ neurons. These data suggest that c-fos mRNA levels are more affected by repeated IMO than corresponding protein, and that arc gene expression does not reflect adaptation in most brain regions, which may be related to its constitutive expression. Therefore, the choice of a particular IEG and the method of measurement are important for proper interpretation of the impact of chronic repeated stress on brain activation.

  3. Immobilization of enzymes by radiation

    International Nuclear Information System (INIS)

    Kaetsu, I.; Kumakura, M.; Yoshida, M.; Asano, M.; Himei, M.; Tamura, M.; Hayashi, K.

    1979-01-01

    Immobilization of various enzymes was performed by radiation-induced polymerization of glass-forming monomers at low temperatures. Alpha-amylase and glucoamylase were effectively immobilized in hydrophilic polymer carrier such as poly(2-hydroxyethyl methacrylate) and also in rather hydrophobic carrier such as poly(tetraethylene-glycol diacrylate). Immobilized human hemoglobin underwent the reversible oxygenation concomitantly with change of oxygen concentration outside of the matrices. (author)

  4. Effects of immobilization on spermiogenesis

    Science.gov (United States)

    Meitner, E. R.

    1980-01-01

    The influence of immobilization stress on spermiogenesis in rats was investigated. After 96 hour immobilization, histological changes began to manifest themselves in the form of practically complete disappearance of cell population of the wall of seminiferous tubule as well as a markedly increased number of cells with pathologic mitoses. Enzymological investigations showed various changes of activity (of acid and alkaline phosphatase and nonspecific esterase) in the 24, 48, and 96 hour immobilization groups.

  5. Biodegradation of chlorobenzene using immobilized crude extracts ...

    African Journals Online (AJOL)

    SERVER

    2007-10-04

    Oct 4, 2007 ... immobilized crude extracts were reused for all other experiments and found that immobilization .... India which are of analytical reagent grade. .... 9. 60. 3. 1. Figure 3. Degradation of chlorobenzene by immobilized crude.

  6. Iodine immobilization in apatites

    International Nuclear Information System (INIS)

    Audubert, F.; Lartigue, J.E.

    2000-01-01

    In the context of a scientific program on long-lived radionuclide conditioning, a matrix for iodine 129 immobilization has been studied. A lead vanado-phosphate apatite was prepared from the melt of lead vanado-phosphate Pb 3 (VO 4 ) 1.6 (PO 4 ) 0.4 and lead iodide PbI 2 in stoichiometric proportions by calcination at 700 deg. C during 3 hours. Natural sintering of this apatite is not possible because the product decomposition occurs at 400 deg. C. Reactive sintering is the solution. The principle depends on the coating of lead iodide with lead vanado-phosphate. Lead vanado-phosphate coating is used as iodo-apatite reactant and as dense covering to confine iodine during synthesis. So the best condition to immobilize iodine during iodo-apatite synthesis is a reactive sintering at 700 deg. C under 25 MPa. We obtained an iodo-apatite surrounded with dense lead vanadate. Leaching behaviour of the matrix synthesized by solid-solid reaction is under progress in order to determine chemical durability, basic mechanisms of the iodo-apatite alteration and kinetic rate law. Iodo-apatite dissolution rates were pH and temperature dependent. We obtained a rate of 2.5 10 -3 g.m -2 .d -1 at 90 deg. C in initially de-ionised water. (authors)

  7. Nucleoside conjugates of quantum dots for characterization of G protein-coupled receptors: strategies for immobilizing A2A adenosine receptor agonists

    Directory of Open Access Journals (Sweden)

    Gao Zhan-Guo

    2010-05-01

    Full Text Available Abstract Background Quantum dots (QDs are crystalline nanoparticles that are compatible with biological systems to provide a chemically and photochemically stable fluorescent label. New ligand probes with fluorescent reporter groups are needed for detection and characterization of G protein-coupled receptors (GPCRs. Results Synthetic strategies for coupling the A2A adenosine receptor (AR agonist CGS21680 (2-[4-(2-carboxyethylphenylethylamino]-5'-N-ethylcarboxamidoadenosine to functionalized QDs were explored. Conjugates tethered through amide-linked chains and poly(ethyleneglycol (PEG displayed low solubility and lacked receptor affinity. The anchor to the dendron was either through two thiol groups of (R-thioctic acid or through amide formation to a commercial carboxy-derivatized QD. The most effective approach was to use polyamidoamine (PAMAM D5 dendrons as multivalent spacer groups, grafted on the QD surface through a thioctic acid moiety. In radioligand binding assays, dendron nucleoside conjugate 11 displayed a moderate affinity at the human A2AAR (Kiapp 1.02 ± 0.15 μM. The QD conjugate of increased water solubility 13, resulting from the anchoring of this dendron derivative, interacted with the receptor with Kiapp of 118 ± 54 nM. The fluorescence emission of 13 occurred at 565 nm, and the presence of the pendant nucleoside did not appreciably quench the fluorescence. Conclusions This is a feasibility study to demonstrate a means of conjugating to a QD a small molecular pharmacophore of a GPCR that is relatively hydrophobic. Further enhancement of affinity by altering the pharmacophore or the linking structures will be needed to make useful affinity probes.

  8. A novel lipid-based nanomicelle of docetaxel: evaluation of antitumor activity and biodistribution

    Directory of Open Access Journals (Sweden)

    Ma M

    2012-07-01

    Full Text Available Mingshu Ma,1 Yanli Hao,1 Nan Liu,1 Zhe Yin,1 Lan Wang,1 Xingjie Liang,2 Xiaoning Zhang11Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing, China; 2National Center for Nanoscience and Technology, Beijing, ChinaPurpose: A lipid-based, nanomicelle-loaded docetaxel (M-DOC was designed and characterized. Optical imaging was employed to evaluate the pharmacokinetics and antitumor efficacy of docetaxel in vivo.Materials and methods: The M-DOC was prepared using the emulsion-diffusion method. Transmission electron microscopy and dynamic light scattering were used to assess the morphology and particle size of the M-DOC. Critical micelle concentrations, their stability under physiological conditions, and their encapsulation efficiency – as measured by high-performance liquid chromatography – were assessed. Pharmacological features were evaluated in two different animal models by comparing M-DOC treatments with docetaxel injections (I-DOC. Bioluminescence imaging was used to assess antitumor activity and docetaxel distribution in vivo, using nude mice injected with luciferase-expressing MDA-MB-231 human breast tumor cells. In addition, animals injected with B16 melanoma cells were used to measure survival time and docetaxel distribution.Results: The M-DOC was prepared as round, uniform spheres with an effective diameter of 20.8 nm. The critical micelle concentration of the original emulsion was 0.06%. Satisfactory encapsulation efficiency (87.6% ± 3.0% and 12-hour stability were achieved. Xenograft results demonstrated that the M-DOC was more effective in inhibiting tumor growth, without significantly changing body weight. Survival was prolonged by 12.6% in the M-DOC group. Tumor growth inhibitory rates in the M-DOC and I-DOC groups were 91.2% and 57.8% in volume and 71.8% and 44.9% in weight, respectively. Optical bioluminescence imaging of tumor growths yielded similar results. Area under the

  9. High-Throughput Lipolysis in 96-Well Plates for Rapid Screening of Lipid-Based Drug Delivery Systems

    DEFF Research Database (Denmark)

    Mosgaard, Mette D; Sassene, Philip J; Mu, Huiling

    2017-01-01

    The high-throughput in vitro intestinal lipolysis model (HTP) applicable for rapid and low-scale screening of lipid-based drug delivery systems (LbDDSs) was optimized and adjusted as to be conducted in 96-well plates (HTP-96). Three different LbDDSs (I-III) loaded with danazol or cinnarizine were...

  10. Assessing attitudes toward spinal immobilization.

    Science.gov (United States)

    Bouland, Andrew J; Jenkins, J Lee; Levy, Matthew J

    2013-10-01

    Prospective studies have improved knowledge of prehospital spinal immobilization. The opinion of Emergency Medical Services (EMS) providers regarding spinal immobilization is unknown, as is their knowledge of recent research advances. To examine the attitudes, knowledge, and comfort of prehospital and Emergency Department (ED) EMS providers regarding spinal immobilization performed under a non-selective protocol. An online survey was conducted from May to July of 2011. Participants were drawn from the Howard County Department of Fire and Rescue Services and the Howard County General Hospital ED. The survey included multiple choice questions and responses on a modified Likert scale. Correlation analysis and descriptive data were used to analyze results. Comfort using the Kendrick Extrication Device was low among ED providers. Experienced providers were more likely to indicate comfort using this device. Respondents often believed that spinal immobilization is appropriate in the management of penetrating trauma to the chest and abdomen. Reported use of padding decreased along with the frequency with which providers practice and encounter immobilized patients. Respondents often indicated that they perform spinal immobilization due solely to mechanism of injury. Providers who feel as if spinal immobilization is often performed unnecessarily were more likely to agree that immobilization causes an unnecessary delay in patient care. The results demonstrate the need for improved EMS education in the use of the Kendrick Extrication Device, backboard padding, and spinal immobilization in the management of penetrating trauma. The attitudes highlighted in this study are relevant to the implementation of a selective spinal immobilization protocol. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Immobilization of Chloroperoxidase on Aminopropyl-Glass

    Science.gov (United States)

    Kadima, Tenshuk A.; Pickard, Michael A.

    1990-01-01

    Chloroperoxidase (CPO) purified from Caldariomyces fumago CMI 89362 was covalently bound to aminopropyl-glass by using a modification of an established method. Acid-washed glass was derivatized by using aminopropyltriethoxysilane, and the enzyme was ionically bound at low ionic strength. Further treatment with glutaraldehyde covalently linked the enzyme to the glass beads in an active form. No elution of bound activity from glass beads could be detected with a variety of washings. The loading of enzyme protein to the glass beads was highest, 100 mg of CPO per g of glass, at high reaction ratios of CPO to glass, but the specific activity of the immobilized enzyme was highest, 36% of theoretical, at low enzyme-to-carrier ratios. No differences in the properties of the soluble and immobilized enzymes could be detected by a number of criteria: their pH-activity and pH-stability profiles were similar, as were their thermal stabilities. After five uses, the immobilized enzyme retained full activity between pH 6.0 and 6.7. PMID:16348352

  12. Oral and transdermal drug delivery systems: role of lipid-based lyotropic liquid crystals.

    Science.gov (United States)

    Rajabalaya, Rajan; Musa, Muhammad Nuh; Kifli, Nurolaini; David, Sheba R

    2017-01-01

    Liquid crystal (LC) dosage forms, particularly those using lipid-based lyotropic LCs (LLCs), have generated considerable interest as potential drug delivery systems. LCs have the physical properties of liquids but retain some of the structural characteristics of crystalline solids. They are compatible with hydrophobic and hydrophilic compounds of many different classes and can protect even biologicals and nucleic acids from degradation. This review, focused on research conducted over the past 5 years, discusses the structural evaluation of LCs and their effects in drug formulations. The structural classification of LLCs into lamellar, hexagonal and micellar cubic phases is described. The structures of these phases are influenced by the addition of surfactants, which include a variety of nontoxic, biodegradable lipids; these also enhance drug solubility. LLC structure influences drug localization, particle size and viscosity, which, in turn, determine drug delivery properties. Through several specific examples, we describe the applications of LLCs in oral and topical drug formulations, the latter including transdermal and ocular delivery. In oral LLC formulations, micelle compositions and the resulting LLC structures can determine drug solubilization and stability as well as intestinal transport and absorption. Similarly, in topical LLC formulations, composition can influence whether the drug is retained in the skin or delivered transdermally. Owing to their enhancement of drug stability and promotion of controlled drug delivery, LLCs are becoming increasingly popular in pharmaceutical formulations.

  13. In-vitro release of diclofenac diethylammonium from lipid-based formulations.

    Science.gov (United States)

    Parsaee, Siamak; Sarbolouki, Mohammad N; Parnianpour, Mohamad

    2002-07-08

    This article presents the preparation and topical performance of some new lipid-based formulations of diclofenac, namely (a) a diclofenac aqueous gel containing mixed micelles (sodium cholate:egg lecithin molar ratio 0.55); (b) diclofenac lotion that contains soya lecithin, ethanol and buffer; and (c) diclofenac lipogel containing egg lecithin, isopropyl myristate, propylene glycol and ethanol. Gel formulations were prepared using Carbomer 934. Release of diclofenac from all formulations was monitored via dialysis through Spectra/por membrane into phosphate buffer (0.2 M pH=7.4) using a Franz cell. Drug release profile and diffusion coefficients were compared with brand formulation (Geigy's Vlotaren Emulgel). Statistical analysis of data show that the diffusion coefficient of the drug from these formulations rank according to the following order: Diclofenac lotion (D=5.308x10(-7) cm(2)/s) >lipogel (D=2.102 x 10(-7) cm(2)/s) >Voltaren Emulgel (1.518 x 10(-7) cm(2)/s) >aqueous gel mixed micelle (0.966 x 10(-7) cm(2)/s). These results show that diclofenac lotion and lipogel maybe more suitable formulations than the conventional topical dosage form.

  14. Lipid Based Formulations of Biopharmaceutics Classification System (BCS Class II Drugs: Strategy, Formulations, Methods and Saturation

    Directory of Open Access Journals (Sweden)

    Šoltýsová I.

    2016-12-01

    Full Text Available Active ingredients in pharmaceuticals differ by their physico-chemical properties and their bioavailability therefore varies. The most frequently used and most convenient way of administration of medicines is oral, however many drugs are little soluble in water. Thus they are not sufficiently effective and suitable for such administration. For this reason a system of lipid based formulations (LBF was developed. Series of formulations were prepared and tested in water and biorelevant media. On the basis of selection criteria, there were selected formulations with the best emulsification potential, good dispersion in the environment and physical stability. Samples of structurally different drugs included in the Class II of the Biopharmaceutics classification system (BCS were obtained, namely Griseofulvin, Glibenclamide, Carbamazepine, Haloperidol, Itraconazol, Triclosan, Praziquantel and Rifaximin, for testing of maximal saturation in formulations prepared from commercially available excipients. Methods were developed for preparation of formulations, observation of emulsification and its description, determination of maximum solubility of drug samples in the respective formulation and subsequent analysis. Saturation of formulations with drugs showed that formulations 80 % XA and 20 % Xh, 35 % XF and 65 % Xh were best able to dissolve the drugs which supports the hypothesis that it is desirable to identify limited series of formulations which could be generally applied for this purpose.

  15. Why Fish Oil Fails: A Comprehensive 21st Century Lipids-Based Physiologic Analysis

    Directory of Open Access Journals (Sweden)

    B. S. Peskin

    2014-01-01

    Full Text Available The medical community suffered three significant fish oil failures/setbacks in 2013. Claims that fish oil’s EPA/DHA would stop the progression of heart disease were crushed when The Risk and Prevention Study Collaborative Group (Italy released a conclusive negative finding regarding fish oil for those patients with high risk factors but no previous myocardial infarction. Fish oil failed in all measures of CVD prevention—both primary and secondary. Another major 2013 setback occurred when fish oil’s DHA was shown to significantly increase prostate cancer in men, in particular, high-grade prostate cancer, in the Selenium and Vitamin E Cancer Prevention Trial (SELECT analysis by Brasky et al. Another monumental failure occurred in 2013 whereby fish oil’s EPA/DHA failed to improve macular degeneration. In 2010, fish oil’s EPA/DHA failed to help Alzheimer’s victims, even those with low DHA levels. These are by no means isolated failures. The promise of fish oil and its so-called active ingredients EPA / DHA fails time and time again in clinical trials. This lipids-based physiologic review will explain precisely why there should have never been expectation for success. This review will focus on underpublicized lipid science with a focus on physiology.

  16. Why fish oil fails: a comprehensive 21st century lipids-based physiologic analysis.

    Science.gov (United States)

    Peskin, B S

    2014-01-01

    The medical community suffered three significant fish oil failures/setbacks in 2013. Claims that fish oil's EPA/DHA would stop the progression of heart disease were crushed when The Risk and Prevention Study Collaborative Group (Italy) released a conclusive negative finding regarding fish oil for those patients with high risk factors but no previous myocardial infarction. Fish oil failed in all measures of CVD prevention-both primary and secondary. Another major 2013 setback occurred when fish oil's DHA was shown to significantly increase prostate cancer in men, in particular, high-grade prostate cancer, in the Selenium and Vitamin E Cancer Prevention Trial (SELECT) analysis by Brasky et al. Another monumental failure occurred in 2013 whereby fish oil's EPA/DHA failed to improve macular degeneration. In 2010, fish oil's EPA/DHA failed to help Alzheimer's victims, even those with low DHA levels. These are by no means isolated failures. The promise of fish oil and its so-called active ingredients EPA / DHA fails time and time again in clinical trials. This lipids-based physiologic review will explain precisely why there should have never been expectation for success. This review will focus on underpublicized lipid science with a focus on physiology.

  17. Spray drying of lipid-based systems loaded with Camellia sinensis polyphenols.

    Science.gov (United States)

    Secolin, Vanessa A; Souza, Claudia R F; Oliveira, Wanderley P

    2017-03-01

    In this work, spray-dried lipid systems based on soy phosphatidylcholine, cholesterol and lauroyl polyoxylglycerides for entrapping Green tea polyphenols were produced. The aim was to study the effects of the encapsulating composition and spray drying conditions on the system performance and physicochemical product properties. The spray dryer powder production yield falls around 50.7 ± 2.8%, which is typical for lab scale spray dryers. Wrinkled and rounded particles, with low surface porosities were generated, independent of the drying carriers (trehalose or lactose) used. The product showed high encapsulation efficiency of Green tea polyphenols, which was promptly redispersible in water. It presented low density, and good compressive and flow properties. The results herein reported confirm the feasibility of the entrapment of Green tea polyphenols in lipid-based compositions by spray drying in presence of the drying carriers evaluated. The spray-dried microparticles show high potential to be used as additive in food, nutraceutical and pharmaceutical products.

  18. Optimization and characterization of stable lipid-based, oxygen-filled microbubbles by mixture design.

    Science.gov (United States)

    Polizzotti, Brian D; Thomson, Lindsay M; O'Connell, Daniel W; McGowan, Francis X; Kheir, John N

    2014-08-01

    Tissue hypoxia is a final common pathway that leads to cellular injury and death in a number of critical illnesses. Intravenous injections of self-assembling, lipid-based oxygen microbubbles (LOMs) can be used to deliver oxygen gas, preventing organ injury and death from systemic hypoxemia. However, current formulations exhibit high polydispersity indices (which may lead to microvascular obstruction) and poor shelf-lives, limiting the translational capacity of LOMs. In this study, we report our efforts to optimize LOM formulations using a mixture response surface methodology (mRSM). We study the effect of changing excipient proportions (the independent variables) on microbubble diameter and product loss (the dependent variables). By using mRSM analysis, the experimental data were fit using a reduced Scheffé linear mixture model. We demonstrate that formulations manufactured from 1,2-distearoyl-sn-glycero-3-phosphocholine, corn syrup, and water produce micron-sized microbubbles with low polydispersity indices, and decreased product loss (relative to previously described formulations) when stored at room temperature over a 30-day period. Optimized LOMs were subsequently tested for their oxygen-releasing ability and found to have similar release kinetics as prior formulations. © 2014 Wiley Periodicals, Inc.

  19. HLW immobilization in glass

    International Nuclear Information System (INIS)

    Leroy, P.; Jacquet-Francillon, N.; Runge, S.

    1992-01-01

    The immobilization of High Level Waste in glass in France is a long history which started as early as in the 1950's. More than 30 years of Research and Development have been invested in that field. Two industrial facilities are operating (AVM and R7) and a third one (T7), under cold testing, is planned to start active operation in the mid-92. While vitrification has been demonstrated to be an industrially mastered process, the question of the quality of the final waste product, i.e. the HLW glass, must be addressed. The scope of the present paper is to focus on the latter point from both standpoints of the R and D and of the industrial reality

  20. [Native, modified, and immobilized chymotrypsin in chaotropic media. Stabilization limits].

    Science.gov (United States)

    Panova, A A; Levitskiĭ, V Iu; Mozhaev, V V

    1994-07-01

    To stabilize alpha-chymotrypsin against irreversible thermal inactivation at high temperatures, methods of covalent modification and multi-point immobilization in combination with the addition of salting-in compounds were used. The upper limit of the protein stability proved to be the same for a combination of the modification and salting-in media and for each of these methods separately. The limit of stabilization reached by means of covalent immobilization is higher than the limit of stabilization reached by two other methods. The greatest stabilization of immobilized alpha-chymotrypsin by the salting-in media (a 10000 fold increase in the native enzyme's stability level) takes place only in the case of the protein with the minimum number of bonds with the support. Stabilization of the enzyme by these methods is explained in terms of the suppression of the conformational inactivation processes.

  1. Catalytic properties of immobilized tannase produced from Aspergillus aculeatus compared with the free enzyme

    Directory of Open Access Journals (Sweden)

    A. B El-Tanash

    2011-09-01

    Full Text Available Aspergillus aculeatus tannase was immobilized on several carriers by entrapment and covalent binding with cross - linking. Tannase immobilized on gelatin with cross - linking agent showed the highest activity and immobilization yield. The optimum pH of the immobilized enzyme was shifted to a more acidic range compared with the free enzyme (from pH 5.5 to pH 5.0. The optimum temperature of the reaction was determined to be 50ºC for the free enzyme and 60ºC for the immobilized form. The thermal stability, as well as stability over a wide range of pH, was significantly improved by the immobilization process. The calculated Km of the immobilized tannase (11.8 mg ml-1 is higher than that of the free tannase (6.5 mg ml-1, while Vmax of the immobilized enzyme (0.32 U (µg protein-1 is lower than that of the free tannase (2.7 U (µg protein-1. The immobilized enzyme was able to retain 84 % of the initial catalytic activity after 5.0 cycles.

  2. MUCOADHESIVE GEL WITH IMMOBILIZED LYSOZYME: PREPARATION AND PROPERTIES

    Directory of Open Access Journals (Sweden)

    Dekina S. S.

    2015-08-01

    Full Text Available The study of non-covalent immobilized lysozyme, as well as physico-chemical and biochemical properties of obtained mucoadhesive gel was the aim of the research. Lysozyme activity was determined by bacteriolytic method (Micrococcus lysodeikticus cells acetone powder was a substrate. Lysozyme immobilization was conducted by the method of entrapment in gel. Enzyme carrier interaction was studied by viscometric, spectrophotometric and spectrofluorimetric methods. Mucoadhesive gel with immobilized lysozyme, possessing antiinflammatory and antimicrobial activities, was prepared. Due to immobilization, protein-polymer complex with the original enzymatic activity was formed. The product is characterized by high mucoadhesive properties, quantitative retaining of protein and bacteriolytic activity, prolonged release of the enzyme, improved biochemical characteristics (extended pH-activity profile, stability in acidic medium and during storage for 2 years, and it is perspective for further studies. The proposed method for lysozyme immobilization in the carboxymethyl cellulose sodium salt gel allows to obtain a stable, highly efficient product, with high adhesive properties for attachment to the mucous membranes, that is promising for use in biomedicine.

  3. Raman Spectroelectrochemical Investigations of Immobilized Redox Proteins

    OpenAIRE

    Grochol, Jana

    2007-01-01

    Das Interesse an auf Elektroden immobilisierten Redoxproteinen ist in den letzten Jahren stark gestiegen. Die oberflächenverstärkte Resonanz-Raman- (SERR) Spektroelektrochemie eignet sich hervorragend zum Studium des heterogenen Elektronentransfers, da sie selektiv das Schwingungsspektrum der Redoxzentren der immobilisierten Spezies abfragt. SERR Spektroskopie kombiniert die Vorteile der Oberflächen- und Resonanz-Verstärkung, indem Hämproteine auf rauen Silberelektroden adsorbiert und in der ...

  4. Visible and UV-curable chitosan derivatives for immobilization of biomolecules.

    Science.gov (United States)

    Kim, Eun-Hye; Han, Ga-Dug; Kim, Jae-Won; Noh, Seung-Hyun; Lee, Jae-Gwan; Ito, Yoshihiro; Son, Tae-Il

    2017-11-01

    Chitosan, which has many biocompatible properties, is used widely in medical field like wound healing, drug delivery and so on. Chitosan could be used as a biomaterial to immobilize protein-drug. There are many methods to immobilize protein-drug, but they have some drawbacks such as low efficiency and denaturation of protein. Therefore, photo-immobilization method is suggested to immobilize protein-drug. Photo-immobilization method is simple-reaction and also needs no additional crosslinking reagent. There has been some effort to modify chitosan to have an ability of photo-immobilization. Generally, visible and UV light reactive chitosan derivatives were prepared. Various types of photo-curable chitosan derivatives showed possibility for application to medical field. For example, they showed ability for protein-immobilization and some of them showed wound-healing effect, anti-adhesive effect, or property to interact directly with titanium surface. In this study, we introduce many types of photo-curable chitosan derivative and their possibility of medical application. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Oral and transdermal drug delivery systems: role of lipid-based lyotropic liquid crystals

    Directory of Open Access Journals (Sweden)

    Rajabalaya R

    2017-02-01

    Full Text Available Rajan Rajabalaya, Muhammad Nuh Musa, Nurolaini Kifli, Sheba R David PAPRSB Institute of Health Sciences, Universiti Brunei Darussalam, Brunei Darussalam Abstract: Liquid crystal (LC dosage forms, particularly those using lipid-based lyotropic LCs (LLCs, have generated considerable interest as potential drug delivery systems. LCs have the physical properties of liquids but retain some of the structural characteristics of crystalline solids. They are compatible with hydrophobic and hydrophilic compounds of many different classes and can protect even biologicals and nucleic acids from degradation. This review, focused on research conducted over the past 5 years, discusses the structural evaluation of LCs and their effects in drug formulations. The structural classification of LLCs into lamellar, hexagonal and micellar cubic phases is described. The structures of these phases are influenced by the addition of surfactants, which include a variety of nontoxic, biodegradable lipids; these also enhance drug solubility. LLC structure influences drug localization, particle size and viscosity, which, in turn, determine drug delivery properties. Through several specific examples, we describe the applications of LLCs in oral and topical drug formulations, the latter including transdermal and ocular delivery. In oral LLC formulations, micelle compositions and the resulting LLC structures can determine drug solubilization and stability as well as intestinal transport and absorption. Similarly, in topical LLC formulations, composition can influence whether the drug is retained in the skin or delivered transdermally. Owing to their enhancement of drug stability and promotion of controlled drug delivery, LLCs are becoming increasingly popular in pharmaceutical formulations. Keywords: liquid crystal, drug delivery, controlled release, lyotropic, surfactants, drug localization

  6. Providing lipid-based nutrient supplements does not affect developmental milestones among Malawian children.

    Science.gov (United States)

    Mangani, Charles; Cheung, Yin Bun; Maleta, Kenneth; Phuka, John; Thakwalakwa, Chrissie; Dewey, Kathryn; Manary, Mark; Puumalainen, Taneli; Ashorn, Per

    2014-01-01

    To assess whether using lipid-based nutrient supplements (LNS) to complement the diets of infants and young children affected when they achieved selected developmental milestones. In rural Malawi, 840 6-month-old healthy infants were enrolled to a randomised trial. Control participants received no supplements, others were provided with milk-containing LNS, soy-containing LNS or corn-soy blend (CSB) for 12 months. Outcomes were the age at which they achieved key milestone: motor (walking with assistance, standing and walking alone, running), social (drinking from a cup and eating by themselves) and language (saying single comprehensible words and waving goodbye). The mean age at which the subjects walked with assistance was 42.5, 42.3, 42.7 and 43.2 weeks in the control, milk-LNS, soy-LNS and CSB groups, respectively (p = 0.748). There were also no significant differences in the mean age at standing alone (45.0, 44.9, 45.1 and 46.3 weeks), walking alone (54.6, 55.1, 55.3, 56.5 weeks), running (64.6, 63.7, 64.8, 65.9 weeks) or any other social or language milestones (each p > 0.10). The findings do not support a hypothesis that providing tested formulations and doses of micronutrient-fortified LNS or CSB would have an impact on when young children in rural Malawi achieved selected developmental milestones. ©2013 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

  7. High-level-waste immobilization

    International Nuclear Information System (INIS)

    Crandall, J.L.

    1982-01-01

    Analysis of risks, environmental effects, process feasibility, and costs for disposal of immobilized high-level wastes in geologic repositories indicates that the disposal system safety has a low sensitivity to the choice of the waste disposal form

  8. The Precipitation Behavior of Poorly Water-Soluble Drugs with an Emphasis on the Digestion of Lipid Based Formulations

    DEFF Research Database (Denmark)

    Khan, Jamal; Rades, Thomas; Boyd, Ben

    2016-01-01

    digestion and drug solubilisation during gastrointestinal transit have been explored in detail, but the implications of drug precipitation beyond the potential adverse effect on bioavailability have received attention only in recent years. Specifically, these implications are that different solid forms...... the events that lead to drug precipitation during the dispersion and digestion of lipid based formulations, common methods used to inhibit precipitation, as well as conventional and newly emerging characterization techniques for studying the solid state form of the precipitated drug. Moreover, selected case...... studies are discussed where drug precipitation has ensued from the digestion of lipid based formulations, as well as the apparent link between drug ionisability and altered solid forms on precipitation, culminating in a discussion about the importance of the solid form on precipitation with relevance...

  9. Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization.

    Science.gov (United States)

    Cowan, Don A; Fernandez-Lafuente, Roberto

    2011-09-10

    The immobilization of proteins (mostly typically enzymes) onto solid supports is mature technology and has been used successfully to enhance biocatalytic processes in a wide range of industrial applications. However, continued developments in immobilization technology have led to more sophisticated and specialized applications of the process. A combination of targeted chemistries, for both the support and the protein, sometimes in combination with additional chemical and/or genetic engineering, has led to the development of methods for the modification of protein functional properties, for enhancing protein stability and for the recovery of specific proteins from complex mixtures. In particular, the development of effective methods for immobilizing large multi-subunit proteins with multiple covalent linkages (multi-point immobilization) has been effective in stabilizing proteins where subunit dissociation is the initial step in enzyme inactivation. In some instances, multiple benefits are achievable in a single process. Here we comprehensively review the literature pertaining to immobilization and chemical modification of different enzyme classes from thermophiles, with emphasis on the chemistries involved and their implications for modification of the enzyme functional properties. We also highlight the potential for synergies in the combined use of immobilization and other chemical modifications. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Computer-aided design of bromelain and papain covalent immobilization

    OpenAIRE

    Bessy Cutiño-Avila; Dayrom Gil Pradas; Carlos Aragón Abreu; Yuniel Fernández Marrero; Martha Hernández de la Torre; Emir Salas Sarduy; María de los Ángeles Chávez Planes; José Manuel Guisán Seijas; Joaquín Díaz Brito; Alberto del Monte-Martínez

    2014-01-01

    Título en español: Diseño asistido por computadora de la inmovilización covalente de bromelina y papaína. Título corto: Computer-aided design of bromelain and papain.  Abstract: Enzymes as immobilized derivatives have been widely used in Food, Agrochemical, Pharmaceutical and Biotechnological industries. Protein immobilization is probably the most used technology to improve the operational stability of these molecules. Bromelain (Ananas comosus) and papain (Carica papaya) are cystein pr...

  11. Status of plutonium ceramic immobilization processes and immobilization forms

    Energy Technology Data Exchange (ETDEWEB)

    Ebbinghaus, B.B.; Van Konynenburg, R.A. [Lawrence Livermore National Lab., CA (United States); Vance, E.R.; Jostsons, A. [Australian Nuclear Science and Technology Organization, Menai (Australia)] [and others

    1996-05-01

    Immobilization in a ceramic followed by permanent emplacement in a repository or borehole is one of the alternatives currently being considered by the Fissile Materials Disposition Program for the ultimate disposal of excess weapons-grade plutonium. To make Pu recovery more difficult, radioactive cesium may also be incorporated into the immobilization form. Valuable data are already available for ceramics form R&D efforts to immobilize high-level and mixed wastes. Ceramics have a high capacity for actinides, cesium, and some neutron absorbers. A unique characteristic of ceramics is the existence of mineral analogues found in nature that have demonstrated actinide immobilization over geologic time periods. The ceramic form currently being considered for plutonium disposition is a synthetic rock (SYNROC) material composed primarily of zirconolite (CaZrTi{sub 2}O{sub 7}), the desired actinide host phase, with lesser amounts of hollandite (BaAl{sub 2}Ti{sub 6}O{sub 16}) and rutile (TiO{sub 2}). Alternative actinide host phases are also being considered. These include pyrochlore (Gd{sub 2}Ti{sub 2}O{sub 7}), zircon (ZrSiO{sub 4}), and monazite (CePO{sub 4}), to name a few of the most promising. R&D activities to address important technical issues are discussed. Primarily these include moderate scale hot press fabrications with plutonium, direct loading of PuO{sub 2} powder, cold press and sinter fabrication methods, and immobilization form formulation issues.

  12. Status of plutonium ceramic immobilization processes and immobilization forms

    International Nuclear Information System (INIS)

    Ebbinghaus, B.B.; Van Konynenburg, R.A.; Vance, E.R.; Jostsons, A.

    1996-01-01

    Immobilization in a ceramic followed by permanent emplacement in a repository or borehole is one of the alternatives currently being considered by the Fissile Materials Disposition Program for the ultimate disposal of excess weapons-grade plutonium. To make Pu recovery more difficult, radioactive cesium may also be incorporated into the immobilization form. Valuable data are already available for ceramics form R ampersand D efforts to immobilize high-level and mixed wastes. Ceramics have a high capacity for actinides, cesium, and some neutron absorbers. A unique characteristic of ceramics is the existence of mineral analogues found in nature that have demonstrated actinide immobilization over geologic time periods. The ceramic form currently being considered for plutonium disposition is a synthetic rock (SYNROC) material composed primarily of zirconolite (CaZrTi 2 O 7 ), the desired actinide host phase, with lesser amounts of hollandite (BaAl 2 Ti 6 O 16 ) and rutile (TiO 2 ). Alternative actinide host phases are also being considered. These include pyrochlore (Gd 2 Ti 2 O 7 ), zircon (ZrSiO 4 ), and monazite (CePO 4 ), to name a few of the most promising. R ampersand D activities to address important technical issues are discussed. Primarily these include moderate scale hot press fabrications with plutonium, direct loading of PuO 2 powder, cold press and sinter fabrication methods, and immobilization form formulation issues

  13. Effects of lipophilic components on the compatibility of lipid-based formulations with hard gelatin capsules.

    Science.gov (United States)

    Chen, Feng-Jing; Etzler, Frank M; Ubben, Johanna; Birch, Amy; Zhong, Li; Schwabe, Robert; Dudhedia, Mayur S

    2010-01-01

    The present study investigated the effect of lipophilic components on the compatibility of propylene glycol (PG)-containing lipid-based drug delivery system (LBDDS) formulations with hard gelatin capsules. The presence of a lipophilic active pharmaceutical ingredient (API) (log P approximately 6.1) and an additional lipophilic excipient (Capmul MCM) significantly affected the activity of PG in the fills and the equilibrium of PG between capsule shells and fills. These changes in activity and equilibrium of PG were furthermore correlated to the mechanical and thermal properties of the liquid-filled capsules and subsequently linked to the shelf-life of the capsules on stability with respect to capsule deformation. The present study also investigated the mechanism by which lipophilic component(s) might affect the activity of PG in the fill formulations and the equilibrium of PG between capsule shells and fills. The activities of PG in two series of "binary" mixtures with Capmul MCM and with Cremophor EL were measured, respectively. The mixtures of PG containing Capmul MCM were found to be more nearly ideal than those containing Cremophor EL. The observed negative deviation from Rauolt's law indicates that the excess free energies of mixing are less then zero indicating favorable interaction between PG and the other component. It is speculated that enhanced hydrogen bonding opportunities with Cremophor EL are responsible for the decreased excess free energy of mixing. Replacement of Cremophor EL with lipophilic API also reduces the hydrogen bonding opportunities for PG in the mixtures. This hypothesis may further explain the increased activity of PG in the fills and the shifted equilibrium of PG toward the capsule shells. Activity determination utilizing headspace gas chromatography (GC) using short 30 min incubation time seems to be a time-efficient approach for assessing capsule-fill compatibility. Direct measurements of PG migration and other physical properties of

  14. In situ ellipsometric study of surface immobilization of flagellar filaments

    Energy Technology Data Exchange (ETDEWEB)

    Kurunczi, S., E-mail: kurunczi@mfa.kfki.hu [Department of Photonics, Research Institute for Technical Physics and Materials Science, H-1121, Konkoly Thege Miklos ut 29-33, Budapest (Hungary); Nemeth, A.; Huelber, T. [Department of Photonics, Research Institute for Technical Physics and Materials Science, H-1121, Konkoly Thege Miklos ut 29-33, Budapest (Hungary); Kozma, P. [Department of Photonics, Research Institute for Technical Physics and Materials Science, H-1121, Konkoly Thege Miklos ut 29-33, Budapest (Hungary); Department of Nanotechnology, Research Institute of Chemical and Process Engineering, Faculty of Information Technology, University of Pannonia, Egyetem u. 10, Veszprem, H-8200 (Hungary); Petrik, P. [Department of Photonics, Research Institute for Technical Physics and Materials Science, H-1121, Konkoly Thege Miklos ut 29-33, Budapest (Hungary); Jankovics, H. [Department of Nanotechnology, Research Institute of Chemical and Process Engineering, Faculty of Information Technology, University of Pannonia, Egyetem u. 10, Veszprem, H-8200 (Hungary); Sebestyen, A. [Department of Photonics, Research Institute for Technical Physics and Materials Science, H-1121, Konkoly Thege Miklos ut 29-33, Budapest (Hungary); Department of Nanotechnology, Research Institute of Chemical and Process Engineering, Faculty of Information Technology, University of Pannonia, Egyetem u. 10, Veszprem, H-8200 (Hungary); Vonderviszt, F. [Department of Photonics, Research Institute for Technical Physics and Materials Science, H-1121, Konkoly Thege Miklos ut 29-33, Budapest (Hungary); Department of Nanotechnology, Research Institute of Chemical and Process Engineering, Faculty of Information Technology, University of Pannonia, Egyetem u. 10, Veszprem, H-8200 (Hungary); Institute of Enzymology, Karolina ut 29-33, Budapest, H-1113 (Hungary); and others

    2010-10-15

    Protein filaments composed of thousands of subunits are promising candidates as sensing elements in biosensors. In this work in situ spectroscopic ellipsometry is applied to monitor the surface immobilization of flagellar filaments. This study is the first step towards the development of layers of filamentous receptors for sensor applications. Surface activation is performed using silanization and a subsequent glutaraldehyde crosslinking. Structure of the flagellar filament layers immobilized on activated and non-activated Si wafer substrates is determined using a two-layer effective medium model that accounted for the vertical density distribution of flagellar filaments with lengths of 300-1500 nm bound to the surface. The formation of the first interface layer can be explained by the multipoint covalent attachment of the filaments, while the second layer is mainly composed of tail pinned filaments floating upwards with the free parts. As confirmed by atomic force microscopy, covalent immobilization resulted in an increased surface density compared to absorption.

  15. Immobilized enzymes: understanding enzyme - surface interactions at the molecular level.

    Science.gov (United States)

    Hoarau, Marie; Badieyan, Somayesadat; Marsh, E Neil G

    2017-11-22

    Enzymes immobilized on solid supports have important and industrial and medical applications. However, their uses are limited by the significant reductions in activity and stability that often accompany the immobilization process. Here we review recent advances in our understanding of the molecular level interactions between proteins and supporting surfaces that contribute to changes in stability and activity. This understanding has been facilitated by the application of various surface-sensitive spectroscopic techniques that allow the structure and orientation of enzymes at the solid/liquid interface to be probed, often with monolayer sensitivity. An appreciation of the molecular interactions between enzyme and surface support has allowed the surface chemistry and method of enzyme attachement to be fine-tuned such that activity and stability can be greatly enhanced. These advances suggest that a much wider variety of enzymes may eventually be amenable to immobilization as green catalysts.

  16. Immobilization needs and technology programs

    International Nuclear Information System (INIS)

    Gray, L.W.; Kan, T.; Shaw, H.; Armantrout, G.

    1995-01-01

    In the aftermath of the Cold War, the US and Russia agreed to large reductions in nuclear weapons. To aid in the selection of long-term management options, DOE has undertaken a multifaceted study to select options for storage and disposition of plutonium in keeping with US policy that plutonium must be subjected to the highest standards of safety, security, and accountability. One alternative being considered is immobilization. To arrive at a suitable immobilization form, we first reviewed published information on high-level waste immobilization technologies and identified 72 possible plutonium immobilization forms to be prescreened. Surviving forms were further screened using multi-attribute utility analysis to determine the most promising technology families. Promising immobilization families were further evaluated to identify chemical, engineering, environmental, safety, and health problems that remain to be solved prior to making technical decisions as to the viability of using the form for long- term disposition of plutonium. From this evaluation, a detailed research and development plan has been developed to provide answers to these remaining questions

  17. Immobilization of cellulase by radiation polymerization

    International Nuclear Information System (INIS)

    Kumakura, M.; Kaetsu, I.

    1983-01-01

    Immobilization of cellulase by radiation polymerization at low temperatures was studied. The enzymatic activity of immobilized cellulase pellets varied with the monomer, enzyme concentration, and the thickness of immobilized cellulase pellets. The optimum monomer concentration in the immobilization of cellulase was 30-50% at the pellet thickness of 1.0 mm, in which the enzymatic activity was 50%. The enzymatic activity of immobilized cellulase pellets was examined using various substrates such as cellobiose, carboxymethylcellulose, and paper pretreated by radiation. It was found that irradiated paper can be hydrolyzed by immobilized cellulase pellets. (author)

  18. Immobile Complex Verbs in Germanic

    DEFF Research Database (Denmark)

    Vikner, Sten

    2005-01-01

    the V° requirements or the V* requirements. Haider (1993, p. 62) and Koopman (1995), who also discuss such immobile verbs, only account for verbs with two prefix-like parts (e.g., German uraufführen ‘to perform (a play) for the first time' or Dutch herinvoeren ‘to reintroduce'), not for the more...... frequent type with only one prefix-like part (e.g., German bauchreden/Dutch buikspreken ‘to ventriloquize'). This analysis will try to account not only for the data discussed in Haider (1993) and Koopman (1995) but also for the following: - why immobile verbs include verbs with only one prefix-like part...... are immobile, - why such verbs are not found in Germanic VO-languages such as English and Scandinavian....

  19. Willingness-to-Pay for Lipid-Based Nutrient Supplements in Burkina Faso

    International Nuclear Information System (INIS)

    Guissou, Rosemonde; Adams, Katherine; Xu Yingying; Vosti, Stephen; Hess, Sonja; Ouedraogo, Jean Bosco

    2014-01-01

    Full text: Supplementing young children’s diets with small-quantity lipid-based nutrient supplements (SQ-LNS) is a promising strategy to prevent growth restriction and improve development. It is uncertain how best to scale up the distribution of SQ-LNS and both private and public approaches are being considered. This paper reports on the willingness-to-pay (WTP) by households in rural Burkina Faso for SQ-LNS designed to prevent childhood undernutrition. In a recent randomized controlled nutrition efficacy trial in the Dandé Health District in rural Burkina Faso, 20 g of SQ-LNS per day along with selected health care services have been shown to increase the linear growth and weight gain of children who received the supplement daily from 9-18 months of age. Given the frequency and duration of prescribed consumption of SQ-LNS (one sachet per day, per child) and inter-household differences in poverty, a hybrid distribution system that reaches target consumers through both public channels and retail markets may be recommended; the viability of the latter will hinge on demand. Therefore, policy makers will need to consider household-level demand for SQ-LNS and the factors that influence demand to guide decisions regarding the cost burden borne by recipients versus public sector programs. Using a contingent valuation approach, we solicited, at baseline and six months later, the hypothetical WTP for a daily dose of 20 g of SQ-LNS and a traditional substitute, herbal teas, from a subsample (N = 319) of households participating in the efficacy trial. WTP for SQ-LNS is positive for almost all households, but WTP for SQ-LNS is, on average, lower than WTP for herbal teas. At baseline, average WTP for a day’s supply of SQ-LNS is $0.22 (4th quarter 2011 USD), while average WTP for herbal teas is $0.28. Approximately six months later, average WTP for LNS and for herbal teas, respectively, are $0.21 and $0.25. These results suggest that while households do value SQ

  20. Oriented Immobilization of His-Tagged Protein on a Redox Active Thiol Derivative of DPTA-Cu(II Layer Deposited on a Gold Electrode—The Base of Electrochemical Biosensors

    Directory of Open Access Journals (Sweden)

    Hanna Radecka

    2013-09-01

    Full Text Available This paper concerns the development of an electrochemical biosensor for the determination of Aβ16–23' and Aβ1–40 peptides. The His-tagged V and VC1 domains of Receptor for Advanced Glycation end Products (RAGE immobilized on a gold electrode surface were used as analytically active molecules. The immobilization of His6–RAGE domains consists of: (i formation of a mixed layer of N-acetylcysteamine (NAC and the thiol derivative of pentetic acid (DPTA; (ii complexation of Cu(II by DPTA; (iii oriented immobilization of His6–RAGE domains via coordination bonds between Cu(II sites from DPTA–Cu(II complex and imidazole nitrogen atoms of a histidine tag. Each modification step was controlled by cyclic voltammetry (CV, Osteryoung square-wave voltammetry (OSWV, and atomic force microscopy (AFM. The applicability of the proposed biosensor was tested in the presence of human plasma, which had no influence on its performance. The detection limits for Aβ1–40 determination were 1.06 nM and 0.80 nM, in the presence of buffer and human plasma, respectively. These values reach the concentration level of Aβ1–40 which is relevant for determination of its soluble form in human plasma, as well as in brain. This indicates the promising future application of biosensor presented for early diagnosis of neurodegenerative diseases.

  1. Immobilization of alpha-amylase from Bacillus circulans GRS 313 on coconut fiber.

    Science.gov (United States)

    Dey, Gargi; Nagpal, Varima; Banerjee, Rintu

    2002-01-01

    A simple and inexpensive method for immobilizing alpha-amylase from Bacillus circulans GRS 313 on coconut fiber was developed. The immobilization conditions for highest efficiency were optimized with respect to immobilization pH of 5.5, 30 degrees C, contact time of 4 h, and enzyme to support a ratio of 1:1 containing 0.12 mg/mL of protein. The catalytic properties of the immobilized enzyme were compared with that of the free enzyme. The activity of amylase adsorbed on coconut fiber was 38.7 U/g of fiber at its optimum pH of 5.7 and 48 degrees C, compared with the maximum activity of 40.2 U/mL of free enzyme at the optimum pH of 4.9 and 48 degrees C. The reutilization capacity of the immobilized enzyme was up to three cycles.

  2. Immobilization of acid digestion residue

    International Nuclear Information System (INIS)

    Greenhalgh, W.O.; Allen, C.R.

    1983-01-01

    Acid digestion treatment of nuclear waste is similar to incineration processes and results in the bulk of the waste being reduced in volume and weight to some residual solids termed residue. The residue is composed of various dispersible solid materials and typically contains the resultant radioactivity from the waste. This report describes the immobilization of the residue in portland cement, borosilicate glass, and some other waste forms. Diagrams showing the cement and glass virtification parameters are included in the report as well as process steps and candidate waste product forms. Cement immobilization is simplest and probably least expensive; glass vitrification exhibits the best overall volume reduction ratio

  3. Recent Advances in Immobilization Strategies for Glycosidases

    Science.gov (United States)

    Karav, Sercan; Cohen, Joshua L.; Barile, Daniela; de Moura Bell, Juliana Maria Leite Nobrega

    2017-01-01

    Glycans play important biological roles in cell-to-cell interactions, protection against pathogens, as well as in proper protein folding and stability, and are thus interesting targets for scientists. Although their mechanisms of action have been widely investigated and hypothesized, their biological functions are not well understood due to the lack of deglycosylation methods for large-scale isolation of these compounds. Isolation of glycans in their native state is crucial for the investigation of their biological functions. However, current enzymatic and chemical deglycosylation techniques require harsh pretreatment and reaction conditions (high temperature and use of detergents) that hinder the isolation of native glycan structures. Indeed, the recent isolation of new endoglycosidases that are able to cleave a wider variety of linkages and efficiently hydrolyze native proteins has opened up the opportunity to elucidate the biological roles of a higher variety of glycans in their native state. As an example, our research group recently isolated a novel Endo-β-N-acetylglucosaminidase from Bifidobacterium longum subsp. infantis ATCC 15697 (EndoBI-1) that cleaves N-N′-diacetyl chitobiose moieties found in the N-linked glycan (N-glycan) core of high mannose, hybrid, and complex N-glycans. This enzyme is also active on native proteins, which enables native glycan isolation, a key advantage when evaluating their biological activities. Efficient, stable, and economically viable enzymatic release of N-glycans requires the selection of appropriate immobilization strategies. In this review, we discuss the state-of-the-art of various immobilization techniques (physical adsorption, covalent binding, aggregation, and entrapment) for glycosidases, as well as their potential substrates and matrices. PMID:27718339

  4. Immobilization of trypsin on sub-micron skeletal polymer monolith

    Energy Technology Data Exchange (ETDEWEB)

    Yao Chunhe [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Graduate School, Chinese Academy of Sciences, Beijing 100049 (China); Qi Li, E-mail: qili@iccas.ac.cn [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Hu Wenbin [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Graduate School, Chinese Academy of Sciences, Beijing 100049 (China); Wang Fuyi [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Yang Gengliang [College of Pharmacy, Hebei University, Baoding 071002 (China)

    2011-04-29

    A new kind of immobilized trypsin reactor based on sub-micron skeletal polymer monolith has been developed. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one- or a multi-step reaction. The proteolytic activity of the immobilized trypsin was measured by monitoring the formation of N-{alpha}-benzoyl-L-arginine (BA) which is the digestion product of a substrate N-{alpha}-benzoyl-L-arginine ethyl ester (BAEE). Results showed that the digestion speed was about 300 times faster than that performed in free solution. The performance of such an enzyme reactor was further demonstrated by digesting protein myoglobin. It has been found that the protein digestion could be achieved in 88 s at 30 deg. C, which is comparable to 24 h digestion in solution at 37 {sup o}C. Furthermore, the immobilized trypsin exhibits increased stability even after continuous use compared to that in free solution. The present monolithic enzyme-reactor provides a promising platform for the proteomic research.

  5. In vivo evaluation of lipid-based formulations for oral delivery of apomorphine and its diester prodrugs

    DEFF Research Database (Denmark)

    Borkar, Nrupa Nitin; Holm, René; Yang, Mingshi

    2016-01-01

    .05) than after DLA-w/o administration, indicating that triglycerides and surfactants improved the oral absorption of DLA. Similarly, Cmax and AUC after dosing apomorphine-o/w were significantly higher (p≤0.05) than that of aqueous suspension. This suggested that lipids and lipolysis products possibly aided......In the present study, the differences in oral absorption of apomorphine and its diester prodrugs and the effect of lipid-based formulations on the absorption of apomorphine or its prodrugs were investigated. Apomorphine, dilauroyl apomorphine (DLA) and dipalmitoyl apomorphine (DPA) were orally...

  6. Radiation immobilization of catalase and its application

    International Nuclear Information System (INIS)

    Wang Guanghui; Ha Hongfei; Wang Xia; Wu Jilan

    1988-01-01

    Catalase was immobilized by a chemical method on porous polyacrylamide particles produced by radiation polymerization of acrylamide monomer at low temperature (-78 0 C). Activity of immobilized catalase was enhanced distinctly by joining a chemical arm to the support. The method of recovery of catalase activity on immobilized polymer was found by soaking it in certain buffer. The treatment of H 2 O 2 both in aqueous solution and alcoholic solution by using the immobilized catalase was performed. (author)

  7. Immobilization and characterization of inulinase from Ulocladium

    Indian Academy of Sciences (India)

    Ulocladium atrum inulinase was immobilized on different composite membranes composed of chitosan/nonwoven fabrics. Km values of free and immobilized U. atrum inulinase on different composite membranes were calculated. The enzyme had optimum pH at 5.6 for free and immobilized U. atrum inulinase on polyester ...

  8. Enhanced activity and stability of L-arabinose isomerase by immobilization on aminopropyl glass.

    Science.gov (United States)

    Zhang, Ye-Wang; Jeya, Marimuthu; Lee, Jung-Kul

    2011-03-01

    Immobilization of Bacillus licheniformis L: -arabinose isomerase (BLAI) on aminopropyl glass modified with glutaraldehyde (4 mg protein g support⁻¹) was found to enhance the enzyme activity. The immobilization yield of BLAI was proportional to the quantity of amino groups on the surface of support. Reducing particle size increased the adsorption capacity (q(m)) and affinity (k(a)). The pH and temperature for immobilization were optimized to be pH 7.1 and 33 °C using response surface methodology (RSM). The immobilized enzyme was characterized and compared to the free enzyme. There is no change in optimal pH and temperature before and after immobilization. However, the immobilized BLAI enzyme achieved 145% of the activity of the free enzyme. Correspondingly, the catalytic efficiency (k(cat)/K(m)) was improved 1.47-fold after immobilization compared to the free enzyme. The thermal stability was improved 138-fold (t₁/₂) increased from 2 to 275 h) at 50 °C following immobilization.

  9. Immobilization of Mitochondria on Graphene

    Science.gov (United States)

    2013-08-29

    poly-L-lysine has also been reported for immobilization of yeast mitochondria. Coating was performed by repetitive washing of cover slips with 0.02...of Poly-L-lysine Applications of PLL PLL is a production of bacterial fermentation and is used as a food preservative. In biology, PLL is used in

  10. IMMOBILIZATION OF ACID PHOSPHATASE (TYPE I) FROM WHEAT GERM ON GLUTARALDEHYDE ACTIVATED CHITOSAN BEADS: OPTIMIZATION AND CHARACTERIZATION

    OpenAIRE

    K. Belho; S.R. Nongpiur; P.K. Ambasht

    2014-01-01

    Acid phosphatase from wheat germ (specific activity 1.327 U/mg protein) was used for immobilization on glutaraldehyde activated chitosan beads. Upon activation of chitosan beads, elongated fibers with pores were observed. The optimum percent immobilization obtained was 81.25%. The pH optimum of immobilized acid phosphatase was 5.5 with a shift of 0.5 units from the pH optimum of soluble enzyme (5.0). The values of Km for p-nitrophenylphosphate with soluble and immobilized acid pho...

  11. End-Point Immobilization of Recombinant Thrombomodulin via Sortase-Mediated Ligation

    Science.gov (United States)

    Jiang, Rui; Weingart, Jacob; Zhang, Hailong; Ma, Yong; Sun, Xue-Long

    2012-01-01

    We report an enzymatic end-point modification and immobilization of recombinant human thrombomodulin (TM), a cofactor for activation of anticoagulant protein C pathway via thrombin. First, a truncated TM mutant consisting of epidermal growth factor-like domains 4–6 (TM456) with a conserved pentapeptide LPETG motif at its C-terminal was expressed and purified in E. coli. Next, the truncated TM456 derivative was site-specifically modified with N-terminal diglycine containing molecules such as biotin and the fluorescent probe dansyl via sortase A (SrtA) mediated ligation (SML). The successful ligations were confirmed by SDS-PAGE and fluorescence imaging. Finally, the truncated TM456 was immobilized onto N-terminal diglycine-functionalized glass slide surface via SML directly. Alternatively, the truncated TM456 was biotinylated via SML and then immobilized onto streptavidin-functionalized glass slide surface indirectly. The successful immobilizations were confirmed by fluorescence imaging. The bioactivity of the immobilized truncated TM456 was further confirmed by protein C activation assay, in which enhanced activation of protein C by immobilized recombinant TM was observed. The sortase A-catalyzed surface ligation took place under mild conditions and is rapid occurring in a single step without prior chemical modification of the target protein. This site-specific covalent modification leads to molecules being arranged in a definitively ordered fashion and facilitating the preservation of the protein’s biological activity. PMID:22372933

  12. Chitin hydrolysis assisted by cell wall degrading enzymes immobilized of Thichoderma asperellum on totally cinnamoylated D-sorbitol beads

    International Nuclear Information System (INIS)

    Fernandes, Kátia F.; Cortijo-Triviño, David; Batista, Karla A.; Ulhoa, Cirano J.; García-Ruiz, Pedro A.

    2013-01-01

    In this study, cell wall degrading enzymes produced by Thrichoderma asperellum (TCWDE) were immobilized on totally cinnamoylated D-sorbitol (TCNSO) beads and used for chitin hydrolysis. In order to optimize immobilization efficiency, the reaction time was varied from 2 to 12 h and reactions were conducted in the presence or absence of Na 2 SO 4 . Immobilized enzymes were analysed concerning to thermal and operational stability. Immobilization in presence of Na 2 SO 4 was 54% more efficient than immobilization in absence of salt. After optimization, 32% of the total enzyme offered was immobilized, with 100% of bounding efficiency, measured as the relation between protein and enzyme immobilized. Free and TCNSO–TCWDE presented very similar kinetics with maximum hydrolysis reached at 90 min of reaction. Thermal stability of both free and TCNSO–TCWDE was similar, with losses in activity after 55 °C. Moreover, free and TCNSO–TCWDE retained 100% activity after 3 h incubation at 55 °C. TCNSO–TCWDE were used in a bath-wise reactor during 14 cycles, producing 1825 μg of N-acetylglucosamine (NAG) maintaining 83% of initial activity. - Highlights: • TCWDE immobilized on TCNSO, a support with highly hydrophobic character • New immobilization strategy for immobilization on a hydrophobic support • TCNSO–TCWDE were retained during washes and during incubation at 55 °C for 3 h

  13. Chitin hydrolysis assisted by cell wall degrading enzymes immobilized of Thichoderma asperellum on totally cinnamoylated D-sorbitol beads

    Energy Technology Data Exchange (ETDEWEB)

    Fernandes, Kátia F., E-mail: katia@icb.ufg.br [Departamento de Bioquímica e Biologia Molecular, Instituo de Ciências Biológicas, Universidade Federal de Goiás, Cx. Postal 131, 74001-970 Goiânia, GO (Brazil); Grupo de Química de Carbohidratos y Biotecnología de Alimentos (QCBA), Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia (Spain); Cortijo-Triviño, David [Grupo de Química de Carbohidratos y Biotecnología de Alimentos (QCBA), Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia (Spain); Batista, Karla A.; Ulhoa, Cirano J. [Departamento de Bioquímica e Biologia Molecular, Instituo de Ciências Biológicas, Universidade Federal de Goiás, Cx. Postal 131, 74001-970 Goiânia, GO (Brazil); García-Ruiz, Pedro A. [Grupo de Química de Carbohidratos y Biotecnología de Alimentos (QCBA), Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia (Spain)

    2013-07-01

    In this study, cell wall degrading enzymes produced by Thrichoderma asperellum (TCWDE) were immobilized on totally cinnamoylated D-sorbitol (TCNSO) beads and used for chitin hydrolysis. In order to optimize immobilization efficiency, the reaction time was varied from 2 to 12 h and reactions were conducted in the presence or absence of Na{sub 2}SO{sub 4}. Immobilized enzymes were analysed concerning to thermal and operational stability. Immobilization in presence of Na{sub 2}SO{sub 4} was 54% more efficient than immobilization in absence of salt. After optimization, 32% of the total enzyme offered was immobilized, with 100% of bounding efficiency, measured as the relation between protein and enzyme immobilized. Free and TCNSO–TCWDE presented very similar kinetics with maximum hydrolysis reached at 90 min of reaction. Thermal stability of both free and TCNSO–TCWDE was similar, with losses in activity after 55 °C. Moreover, free and TCNSO–TCWDE retained 100% activity after 3 h incubation at 55 °C. TCNSO–TCWDE were used in a bath-wise reactor during 14 cycles, producing 1825 μg of N-acetylglucosamine (NAG) maintaining 83% of initial activity. - Highlights: • TCWDE immobilized on TCNSO, a support with highly hydrophobic character • New immobilization strategy for immobilization on a hydrophobic support • TCNSO–TCWDE were retained during washes and during incubation at 55 °C for 3 h.

  14. The Precipitation Behavior of Poorly Water-Soluble Drugs with an Emphasis on the Digestion of Lipid Based Formulations.

    Science.gov (United States)

    Khan, Jamal; Rades, Thomas; Boyd, Ben

    2016-03-01

    An increasing number of newly discovered drugs are poorly water-soluble and the use of natural and synthetic lipids to improve the oral bioavailability of these drugs by utilizing the digestion pathway in-vivo has proved an effective formulation strategy. The mechanisms responsible for lipid digestion and drug solubilisation during gastrointestinal transit have been explored in detail, but the implications of drug precipitation beyond the potential adverse effect on bioavailability have received attention only in recent years. Specifically, these implications are that different solid forms of drug on precipitation may affect the total amount of drug absorbed in-vivo through their different physico-chemical properties, and the possibility that the dynamic environment of the small intestine may afford re-dissolution of precipitated drug if present in a high-energy form. This review describes the events that lead to drug precipitation during the dispersion and digestion of lipid based formulations, common methods used to inhibit precipitation, as well as conventional and newly emerging characterization techniques for studying the solid state form of the precipitated drug. Moreover, selected case studies are discussed where drug precipitation has ensued from the digestion of lipid based formulations, as well as the apparent link between drug ionisability and altered solid forms on precipitation, culminating in a discussion about the importance of the solid form on precipitation with relevance to the total drug absorbed.

  15. Protective Effect of a Lipid-Based Preparation from Mycobacterium smegmatis in a Murine Model of Progressive Pulmonary Tuberculosis

    Directory of Open Access Journals (Sweden)

    Maria de los Angeles García

    2014-01-01

    Full Text Available A more effective vaccine against tuberculosis (TB is urgently needed. Based on its high genetic homology with Mycobacterium tuberculosis (Mtb, the nonpathogenic mycobacteria, Mycobacterium smegmatis (Ms, could be an attractive source of potential antigens to be included in such a vaccine. We evaluated the capability of lipid-based preparations obtained from Ms to provide a protective response in Balb/c mice after challenge with Mtb H37Rv strain. The intratracheal model of progressive pulmonary TB was used to assess the level of protection in terms of bacterial load as well as the pathological changes in the lungs of immunized Balb/c mice following challenge with Mtb. Mice immunized with the lipid-based preparation from Ms either adjuvanted with Alum (LMs-AL or nonadjuvanted (LMs showed significant reductions in bacterial load (P<0.01 compared to the negative control group (animals immunized with phosphate buffered saline (PBS. Both lipid formulations showed the same level of protection as Bacille Calmette and Guerin (BCG. Regarding the pathologic changes in the lungs, mice immunized with both lipid formulations showed less pneumonic area when compared with the PBS group (P<0.01 and showed similar results compared with the BCG group. These findings suggest the potential of LMs as a promising vaccine candidate against TB.

  16. AMP kinase expression and activity in human skeletal muscle: effects of immobilization, retraining, and creatine supplementation

    DEFF Research Database (Denmark)

    Eijnde, Bert O.; Derave, Wim; Wojtaszewski, Jørgen

    2005-01-01

    The effects of leg immobilization and retraining in combination with oral creatine intake on muscle AMP-activated protein kinase (AMPK) protein expression and phosphorylation status were investigated. A double-blind trial was performed in young healthy volunteers (n = 22). A cast immobilized...... the right leg for 2 wk, whereafter the knee-extensor muscles of that leg were retrained for 6 wk. Half of the subjects received creatine monohydrate throughout the study (Cr; from 15 g down to 2.5 g daily), and the others ingested placebo (P; maltodextrin). Before and after immobilization and retraining...... that immobilization-induced muscle inactivity for 2 wk does not alter AMPK a1-, a2-, and ß2-subunit expression or a-AMPK phosphorylation status. Furthermore, the present observations indicate that AMPK probably is not implicated in the previously reported beneficial effects of oral creatine supplementation on muscle...

  17. Characterization of immobilization methods of antiviral antibodies in serum for electrochemical biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Huy, Tran Quang, E-mail: huytq@nihe.org.vn [National Institute of Hygiene and Epidemiology (NIHE), No1 Yersin St., Hanoi (Viet Nam); International Training Institute for Materials Science (ITIMS), Hanoi University of Science and Technology (HUST), No1 Dai Co Viet, Hanoi (Viet Nam); Hanh, Nguyen Thi Hong; Van Chung, Pham; Anh, Dang Duc; Nga, Phan Thi [National Institute of Hygiene and Epidemiology (NIHE), No1 Yersin St., Hanoi (Viet Nam); Tuan, Mai Anh, E-mail: tuanma-itims@mail.hut.edu.vn [International Training Institute for Materials Science (ITIMS), Hanoi University of Science and Technology (HUST), No1 Dai Co Viet, Hanoi (Viet Nam)

    2011-06-01

    In this paper, we describes different methods to immobilize Japanese encephalitis virus (JEV) antibodies in human serum onto the interdigitated surface of a microelectrode sensor for optimizing electrochemical detection: (1) direct covalent binding to the silanized surface, (2) binding to the silanized surface via a cross-linker of glutaraldehyde (GA), (3) binding to glutaraldehyde/silanized surface via goat anti-human IgG polyclonal antibody and (4) binding to glutaraldehyde/silanized surface via protein A (PrA). Field emission scanning electron microscopy, Fourier transform infrared spectrometry, and fluorescence microscopy are used to verify the characteristics of antibodies on the interdigitated surface after the serum antibodies immobilization. The analyzed results indicate that the use of protein A is an effective choice for immobilization and orientation of antibodies in serum for electrochemical biosensors. This study provides an advantageous immobilization method of serum containing antiviral antibodies to develop electrochemical biosensors for preliminary screening of viruses in clinical samples from outbreaks.

  18. Biodiesel production with immobilized lipase: A review.

    Science.gov (United States)

    Tan, Tianwei; Lu, Jike; Nie, Kaili; Deng, Li; Wang, Fang

    2010-01-01

    Fatty acid alkyl esters, also called biodiesel, are environmentally friendly and show great potential as an alternative liquid fuel. Biodiesel is produced by transesterification of oils or fats with chemical catalysts or lipase. Immobilized lipase as the biocatalyst draws high attention because that process is "greener". This article reviews the current status of biodiesel production with immobilized lipase, including various lipases, immobilization methods, various feedstocks, lipase inactivation caused by short chain alcohols and large scale industrialization. Adsorption is still the most widely employed method for lipase immobilization. There are two kinds of lipase used most frequently especially for large scale industrialization. One is Candida antartica lipase immobilized on acrylic resin, and the other is Candida sp. 99-125 lipase immobilized on inexpensive textile membranes. However, to further reduce the cost of biodiesel production, new immobilization techniques with higher activity and stability still need to be explored. Copyright 2010 Elsevier Inc. All rights reserved.

  19. Co-immobilized Coupled Enzyme Systems in Biotechnology

    Science.gov (United States)

    2010-01-01

    coimmobilized by ~n­ capsulation in silica spheres that were formed by a polymer -templated silicificatiOn reaction (Betancor et al., 2006). Nitrobenzene...F. , FERNANDEZ-LAFUENTE, R. , GUISAN J. M. (2005). Stabilization of enzymes by multipoint immobilization of thiolated proteins on new epoxy-thiol... polymer monoliths in microftuidic devices for steady- state kinetic analysis and spatially separated multi-enzyme reactions. Analytical Chemistry, 79

  20. Immobilization/remobilization and the regulation of muscle mass

    Science.gov (United States)

    Almon, R. R.

    1983-01-01

    The relationship between animal body weight and the wet and dry weights of the soleus and EDL muscles was derived. Procedures were examined for tissue homogenization, fractionation, protein determination and DNA determination. A sequence of procedures and buffers were developed to carry out all analyses on one small muscle. This would yield a considerable increase in analytical strength associated with paired statistics. The proposed casting procedure which was to be used for immobilization was reexamined.

  1. Magnetic nanoparticles coated with polyaniline to stabilize immobilized trypsin

    Energy Technology Data Exchange (ETDEWEB)

    Maciel, J. C., E-mail: jackeline-maciel@hotmail.com [Universidade Federal de Roraima (Brazil); Mercês, A. A. D.; Cabrera, M. [Universidade Federal de Pernambuco, Laboratório de Imunopatologia Keizo Asami (Brazil); Shigeyosi, W. T. [Universidade Federal de São Carlos, Departamento de Física (Brazil); Souza, S. D. de; Olzon-Dionysio, M.; Fabris, J. D. [Universidade Federal dos Vales de Jequitinhonha e Mucuri (Brazil); Cardoso, C. A. [Universidade Federal de São Carlos, Departamento de Física (Brazil); Neri, D. F. M. [Universidade Federal do Vale do São Francisco (Brazil); Silva, M. P. C.; Carvalho, L. B. [Universidade Federal de Pernambuco, Laboratório de Imunopatologia Keizo Asami (Brazil)

    2016-12-15

    It is reported the synthesis of magnetic nanoparticles via the chemical co-precipitation of Fe {sup 3+} ions and their preparation by coating them with polyaniline. The electronic micrograph analysis showed that the mean diameter for the nanoparticles is ∼15 nm. FTIR, powder X-ray diffraction and Mössbauer spectroscopy were used to understand the chemical, crystallographic and {sup 57}Fe hyperfine structures for the two samples. The nanoparticles, which exhibited magnetic behavior with relatively high spontaneous magnetization at room temperature, were identified as being mainly formed by maghemite (γFe{sub 2}O{sub 3}). The coated magnetic nanoparticles (sample labeled “mPANI”) presented a real ability to bind biological molecules such as trypsin, forming the magnetic enzyme derivative (sample “mPANIG-Trypsin”). The amount of protein and specific activity of the immobilized trypsin were found to be 13±5 μg of protein/mg of mPANI (49.3 % of immobilized protein) and 24.1±0.7 U/mg of immobilized protein, respectively. After 48 days of storage at 4 {sup ∘}C, the activity of the immobilized trypsin was found to be 89 % of its initial activity. This simple, fast and low-cost procedure was revealed to be a promising way to prepare mPANI nanoparticles if technological applications addressed to covalently link biomolecules are envisaged. This route yields chemically stable derivatives, which can be easily recovered from the reaction mixture with a magnetic field and recyclable reused.

  2. Immobilization of Glucose Oxidase to Nanostructured Films of Polystyrene-block-poly(2-vinylpyridine)

    OpenAIRE

    Bhakta, Samir A; Benavidez, Tomas E; Garcia, Carlos D

    2014-01-01

    A critical step for the development of biosensors is the immobilization of the biorecognition element to the surface of a substrate. Among other materials that can be used as substrates, block copolymers have the untapped potential to provide significant advantages for the immobilization of proteins. To explore such possibility, this manuscript describes the fabrication and characterization of thin-films of polystyrene-block-poly(2-vinylpyridine) (PS-b-P2VP). These films were then used to inv...

  3. Delayed Recovery of Skeletal Muscle Mass following Hindlimb Immobilization in mTOR Heterozygous Mice

    OpenAIRE

    Lang, Susan M.; Kazi, Abid A.; Hong-Brown, Ly; Lang, Charles H.

    2012-01-01

    The present study addressed the hypothesis that reducing mTOR, as seen in mTOR heterozygous (+/-) mice, would exaggerate the changes in protein synthesis and degradation observed during hindlimb immobilization as well as impair normal muscle regrowth during the recovery period. Atrophy was produced by unilateral hindlimb immobilization and data compared to the contralateral gastrocnemius. In wild-type (WT) mice, the gradual loss of muscle mass plateaued by day 7. This response was associated ...

  4. Poly(acrylonitrile)chitosan composite membranes for urease immobilization.

    Science.gov (United States)

    Gabrovska, Katya; Georgieva, Aneliya; Godjevargova, Tzonka; Stoilova, Olya; Manolova, Nevena

    2007-05-10

    (Poly)acrylonitrile/chitosan (PANCHI) composite membranes were prepared. The chitosan layer was deposited on the surface as well as on the pore walls of the base membrane. This resulted in the reduction of the pore size of the membrane and in an increase of their hydrophilicity. The pore structure of PAN and PANCHI membranes were determined by TEM and SEM analyses. It was found that the average size of the pore under a selective layer base PAN membrane is 7 microm, while the membrane coated with 0.25% chitosan shows a reduced pore size--small or equal to 5 microm and with 0.35% chitosan--about 4 microm. The amounts of the functional groups, the degree of hydrophilicity and transport characteristics of PAN/Chitosan composite membranes were determined. Urease was covalently immobilized onto all kinds of PAN/chitosan composite membranes using glutaraldehyde. Both the amount of bound protein and relative activity of immobilized urease were measured. The highest activity (94%) was measured for urease bound to PANCHI2 membranes (0.25% chitosan). The basic characteristics (pH(opt), pH(stability), T(opt), T(stability), heat inactivation and storage stability) of immobilized urease were determined. The obtained results show that the poly(acrylonitrile)chitosan composite membranes are suitable for enzyme immobilization.

  5. Polyketone polymer: a new support for direct enzyme immobilization.

    Science.gov (United States)

    Agostinelli, E; Belli, F; Tempera, G; Mura, A; Floris, G; Toniolo, L; Vavasori, A; Fabris, S; Momo, F; Stevanato, R

    2007-01-20

    Polyketone polymer -[-CO-CH(2)-CH(2)-](n)-, obtained by copolymerization of ethene and carbon monoxide, is utilized for immobilization of three different enzymes, one peroxidase from horseradish (HRP) and two amine oxidases, from bovine serum (BSAO) and lentil seedlings (LSAO). The easy immobilization procedure is carried out in diluted buffer, at pH 7.0 and 3 degrees C, gently mixing the proteins with the polymer. No bifunctional reagents and spacer arms are required for the immobilization, which occurs exclusively via a large number of hydrogen bonds between the carbonyl groups of the polymer and the -NH groups of the polypeptidic chain. Experiments demonstrate a high linking capacity of polymer for BSAO and an extraordinary strong linkage for LSAO. Moreover, activity measurements demonstrate that immobilized LSAO totally retains the catalytic characteristics of the free enzyme, where only a limited increase of K(M) value is observed. Finally, the HRP-activated polymer is successfully used as active packed bed of an enzymatic reactor for continuous flow conversion and flow injection analysis of hydrogen peroxide containing solutions.

  6. Immobilization of iodine in concrete

    Science.gov (United States)

    Clark, Walter E.; Thompson, Clarence T.

    1977-04-12

    A method for immobilizing fission product radioactive iodine recovered from irradiated nuclear fuel comprises combining material comprising water, Portland cement and about 3-20 wt. % iodine as Ba(IO.sub.3).sub.2 to provide a fluid mixture and allowing the fluid mixture to harden, said Ba(IO.sub.3).sub.2 comprising said radioactive iodine. An article for solid waste disposal comprises concrete prepared by this method. BACKGROUND OF THE INVENTION This invention was made in the course of, or under a contract with the Energy Research and Development Administration. It relates in general to reactor waste solidification and more specifically to the immobilization of fission product radioactive iodine recovered from irradiated nuclear fuel for underground storage.

  7. Contaminant immobilization via microbial activity

    International Nuclear Information System (INIS)

    1991-11-01

    The aim of this study was to search the literature to identify biological techniques that could be applied to the restoration of contaminated groundwaters near uranium milling sites. Through bioremediation it was hypothesized that the hazardous heavy metals could be immobilized in a stable, low-solubility form, thereby halting their progress in the migrating groundwater. Three basic mechanisms were examined: reduction of heavy metals by microbially produced hydrogen sulfide; direct microbial mediated reduction; and biosorption

  8. Immobilization of iodine in concrete

    International Nuclear Information System (INIS)

    Clark, W.E.; Thompson, C.T.

    1977-01-01

    A method for immobilizing fission product radioactive iodine recovered from irradiated nuclear fuel comprises combining material comprising water, Portland cement and about 3 to 20 wt percent iodine as Ba(IO 3 ) 2 to provide a fluid mixture and allowing the fluid mixture to harden, said Ba(IO 3 ) 2 comprising said radioactive iodine. An article for solid waste disposal comprises concrete prepared by this method. 10 claims, 2 figures

  9. Immobilization and continuous culture of cells with radiation polymerized supports for the uses of biomass conversion processes

    International Nuclear Information System (INIS)

    Kaetsu, I.; Kumakura, M.; Fujimura, T.; Tamada, M.; Kasai, N.

    1988-01-01

    A novel technique for immobilization and biofunctional components such as enzyme, antibody, protein, drug, hormone and organella by means of radiation polymerization was studied and developed. (E.G.) [pt

  10. Lipid-based nutrient supplements do not affect efavirenz but lower plasma nevirapine concentrations in Ethiopian adult HIV patients

    DEFF Research Database (Denmark)

    Abdissa, A; Olsen, Mette Frahm; Yilma, D

    2015-01-01

    OBJECTIVES: Lipid-based nutrient supplements (LNSs) are increasingly used in HIV programmes in resource-limited settings. However, the possible effects of LNSs on the plasma concentrations of antiretroviral drugs have not been assessed. Here, we aimed to assess the effects of LNSs on plasma...... efavirenz and nevirapine trough concentrations in Ethiopian adult HIV-infected patients. METHODS: The effects of LNSs were studied in adults initiating antiretroviral therapy (ART) in a randomized trial. Patients with body mass index (BMI) > 17 kg/m(2) (n = 282) received daily supplementation of an LNS.......9; -0.9 μg/mL; P = 0.01), respectively, compared with the group not receiving supplements. There were no differences between groups with respect to efavirenz plasma concentrations. The CYP2B6 516 G>T polymorphism was associated with a 5 μg/mL higher plasma efavirenz concentration compared with the wild...

  11. Improvement of enantioselectivity by immobilized imprinting of epoxide hydrolase from Rhodotorula glutinis

    NARCIS (Netherlands)

    Kronenburg, N.A.E.; Bont, de J.A.M.; Fischer, L.

    2001-01-01

    The yeast Rhodotorula glutinis contains an enantioselective, membrane-associated epoxide hydrolase (EH). Partially purified EH was immobilized in a two-step procedure. In the first step, the proteins were derivatized with itaconic anhydride. In the second step, the derivatized proteins were

  12. Phase Transitions of Isotropic to Anisotropic Biocompatible Lipid-Based Drug Delivery Systems Overcoming Insoluble Benznidazole Loading

    Science.gov (United States)

    Streck, Letícia; Sarmento, Víctor H. V.; Machado, Paula R. L.; Farias, Kleber J. S.; Fernandes-Pedrosa, Matheus F.; da Silva-Júnior, Arnóbio Antônio

    2016-01-01

    Previous studies reported low benznidazole (BNZ) loading in conventional emulsions due to the weak interaction of the drug with the most common oils used to produce foods or pharmaceuticals. In this study, we focused on how the type of surfactant, surfactant-to-oil ratio w/w (SOR) and oil-to-water ratio w/w (OWR) change the phase behavior of different lipid-based drug delivery systems (LBDDS) produced by emulsion phase inversion. The surfactant mixture composed of soy phosphatidylcholine and sodium oleate (1:7, w/w, hydrophilic lipophilic balance = 16) stabilized medium chain triglyceride in water. Ten formulations with the clear aspect or less turbid dispersions (five with the SOR ranging from 0.5 to 2.5 and five with the OWR from 0.06 to 0.4) were selected from the phase behavior diagram to assess structural features and drug-loading capacity. The rise in the SOR induced the formation of distinct lipid-based drug delivery systems (nanoemulsions and liquid crystal lamellar type) that were identified using rheological measurements and cross-polarized light microscopy images. Clear dispersions of small and narrow droplet-sized liquid-like nanoemulsions, Newtonian flow-type, were produced at SOR from 0.5 to 1.5 and OWR from 0.12 to 0.4, while clear liquid or gel-like liquid crystals were produced at SOR from 1.5 to 2.5. The BNZ loading was improved according to the composition and type of LBDDS produced, suggesting possible drug location among surfactant layers. The cell viability assays proved the biocompatibility for all of the prepared nanoemulsions at SOR less than 1.5 and liquid crystals at SOR less than 2.5, demonstrating their promising features for the oral or parenteral colloidal delivery systems containing benznidazole for Chagas disease treatment. PMID:27376278

  13. Cationic lipid-based nanoparticles mediate functional delivery of acetate to tumor cells in vivo leading to significant anticancer effects

    Directory of Open Access Journals (Sweden)

    Brody LP

    2017-09-01

    Full Text Available Leigh P Brody,1,* Meliz Sahuri-Arisoylu,1,* James R Parkinson,1 Harry G Parkes,2 Po Wah So,3 Nabil Hajji,4 E Louise Thomas,1 Gary S Frost,5 Andrew D Miller,6,* Jimmy D Bell1,* 1Department of Life Sciences, Faculty of Science and Technology, University of Westminster, 2CR-UK Clinical MR Research Group, Institute of Cancer Research, Sutton, Surrey, 3Department of Neuroimaging, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, 4Department of Medicine, Division of Experimental Medicine, Centre for Pharmacology & Therapeutics, Toxicology Unit, Imperial College London, 5Faculty of Medicine, Nutrition and Dietetic Research Group, Division of Diabetes, Endocrinology and Metabolism, Department of Investigative Medicine, Imperial College London, Hammersmith Hospital, 6Institute of Pharmaceutical Science, King’s College London, London, UK *These authors contributed equally to this work Abstract: Metabolic reengineering using nanoparticle delivery represents an innovative therapeutic approach to normalizing the deregulation of cellular metabolism underlying many diseases, including cancer. Here, we demonstrated a unique and novel application to the treatment of malignancy using a short-chain fatty acid (SCFA-encapsulated lipid-based delivery system – liposome-encapsulated acetate nanoparticles for cancer applications (LITA-CAN. We assessed chronic in vivo administration of our nanoparticle in three separate murine models of colorectal cancer. We demonstrated a substantial reduction in tumor growth in the xenograft model of colorectal cancer cell lines HT-29, HCT-116 p53+/+ and HCT-116 p53-/-. Nanoparticle-induced reductions in histone deacetylase gene expression indicated a potential mechanism for these anti-proliferative effects. Together, these results indicated that LITA-CAN could be used as an effective direct or adjunct therapy to treat malignant transformation in vivo. Keywords: lipid-based nanoparticles, liposomes

  14. Phase Transitions of Isotropic to Anisotropic Biocompatible Lipid-Based Drug Delivery Systems Overcoming Insoluble Benznidazole Loading

    Directory of Open Access Journals (Sweden)

    Letícia Streck

    2016-06-01

    Full Text Available Previous studies reported low benznidazole (BNZ loading in conventional emulsions due to the weak interaction of the drug with the most common oils used to produce foods or pharmaceuticals. In this study, we focused on how the type of surfactant, surfactant-to-oil ratio w/w (SOR and oil-to-water ratio w/w (OWR change the phase behavior of different lipid-based drug delivery systems (LBDDS produced by emulsion phase inversion. The surfactant mixture composed of soy phosphatidylcholine and sodium oleate (1:7, w/w, hydrophilic lipophilic balance = 16 stabilized medium chain triglyceride in water. Ten formulations with the clear aspect or less turbid dispersions (five with the SOR ranging from 0.5 to 2.5 and five with the OWR from 0.06 to 0.4 were selected from the phase behavior diagram to assess structural features and drug-loading capacity. The rise in the SOR induced the formation of distinct lipid-based drug delivery systems (nanoemulsions and liquid crystal lamellar type that were identified using rheological measurements and cross-polarized light microscopy images. Clear dispersions of small and narrow droplet-sized liquid-like nanoemulsions, Newtonian flow-type, were produced at SOR from 0.5 to 1.5 and OWR from 0.12 to 0.4, while clear liquid or gel-like liquid crystals were produced at SOR from 1.5 to 2.5. The BNZ loading was improved according to the composition and type of LBDDS produced, suggesting possible drug location among surfactant layers. The cell viability assays proved the biocompatibility for all of the prepared nanoemulsions at SOR less than 1.5 and liquid crystals at SOR less than 2.5, demonstrating their promising features for the oral or parenteral colloidal delivery systems containing benznidazole for Chagas disease treatment.

  15. Immobilization and kinetics of catalase on calcium carbonate nanoparticles attached epoxy support.

    Science.gov (United States)

    Preety; Hooda, Vinita

    2014-01-01

    A novel hybrid epoxy/nano CaCO3 composite matrix for catalase immobilization was prepared by polymerizing epoxy resin in the presence of CaCO3 nanoparticles. The hybrid support was characterized using scanning electron microscopy and Fourier transform infrared spectroscopy. Catalase was successfully immobilized onto epoxy/nano CaCO3 support with a conjugation yield of 0.67 ± 0.01 mg/cm(2) and 92.63 ± 0.80 % retention of activity. Optimum pH and optimum temperature of free and immobilized catalases were found to be 7.0 and 35 °C. The value of Km for H2O2 was higher for immobilized enzyme (31.42 mM) than native enzyme (27.73 mM). A decrease in Vmax value from 1,500 to 421.10 μmol (min mg protein)(-1) was observed after immobilization. Thermal and storage stabilities of catalase improved immensely after immobilization. Immobilized enzyme retained three times than the activity of free enzyme when kept at 75 °C for 1 h and the half-life of enzyme increased five times when stored in phosphate buffer (0.01 M, pH 7.0) at 5 °C. The enzyme could be reused 30 times without any significant loss of its initial activity. Desorption of catalase from the hybrid support was minimum at pH 7.0.

  16. Complementary feeding messages that target cultural barriers enhance both the use of lipid-based nutrient supplements and underlying feeding practices to improve infant diets in rural Zimbabwe.

    Science.gov (United States)

    Paul, Keriann H; Muti, Monica; Chasekwa, Bernard; Mbuya, Mduduzi N N; Madzima, Rufaro C; Humphrey, Jean H; Stoltzfus, Rebecca J

    2012-04-01

    Supplementation with lipid-based nutrient supplements (LiNS) is promoted as an approach to prevent child undernutrition and growth faltering. Previous LiNS studies have not tested the effects of improving the underlying diet prior to providing LiNS. Formative research was conducted in rural Zimbabwe to develop feeding messages to improve complementary feeding with and without LiNS. Two rounds of Trials of Improved Practices were conducted with mothers of infants aged 6-12 months to assess the feasibility of improving infant diets using (1) only locally available resources and (2) locally available resources plus 20 g of LiNS as Nutributter®/day. Common feeding problems were poor dietary diversity and low energy density. Popular improved practices were to process locally available foods so that infants could swallow them and add processed local foods to enrich porridges. Consumption of beans, fruits, green leafy vegetables, and peanut/seed butters increased after counselling (P < 0.05). Intakes of energy, protein, vitamin A, folate, calcium, iron and zinc from complementary foods increased significantly after counselling with or without the provision of Nutributter (P < 0.05). Intakes of fat, folate, iron, and zinc increased only (fat) or more so (folate, iron, and zinc) with the provision of Nutributter (P < 0.05). While provision of LiNS was crucial to ensure adequate intakes of iron and zinc, educational messages that were barrier-specific and delivered directly to mothers were crucial to improving the underlying diet. © 2010 Blackwell Publishing Ltd.

  17. Influence of chirality on catalytic generation of nitric oxide and platelet behavior on selenocystine immobilized TiO2 films.

    Science.gov (United States)

    Fan, Yonghong; Pan, Xiaxin; Wang, Ke; Wu, Sisi; Han, Honghong; Yang, Ping; Luo, Rifang; Wang, Hong; Huang, Nan; Tan, Wei; Weng, Yajun

    2016-09-01

    As nitric oxide (NO) plays vital roles in the cardiovascular system, incorporating this molecule into cardiovascular stents is considered as an effective method. In the present study, selenocystine with different chirality (i.e., l- and d-selenocystine) was used as the catalytic molecule immobilized on TiO2 films for decomposing endogenous NO donor. The influences of surface chirality on NO release and platelet behavior were evaluated. Results show that although the amount of immobilized l-selenocystine on the surface was nearly the same as that of immobilized d-selenocystine, in vitro catalytic NO release tests showed that l-selenocystine immobilized surfaces were more capable of catalyzing the decomposition of S-nitrosoglutathione and thus generating more NO. Accordingly, l-selenocystine immobilized surfaces demonstrated significantly increased inhibiting effects on the platelet adhesion and activation, when compared to d-selenocystine immobilized ones. Measurement of the cGMP concentration of platelets further confirmed that surface chirality played an important role in regulating NO generation and platelet behaviors. Additionally, using bovine serum albumin and fibrinogen as model proteins, the protein adsorption determined with quartz crystal microbalance showed that the l-selenocystine immobilized surface enhanced protein adsorption. In conclusion, surface chirality significantly influences protein adsorption and NO release, which may have significant implications in the design of NO-generating cardiovascular stents. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Effect of hydrogel elasticity and ephrinB2-immobilized manner on Runx2 expression of human mesenchymal stem cells.

    Science.gov (United States)

    Toda, Hiroyuki; Yamamoto, Masaya; Uyama, Hiroshi; Tabata, Yasuhiko

    2017-08-01

    The objective of this study is to design the manner of ephrinB2 immobilized onto polyacrylamide (PAAm) hydrogels with varied elasticity and evaluate the effect of hydrogels elasticity and the immobilized manner of ephrinB2 on the Runx2 expression of human mesenchymal stem cells (hMSC). The PAAm hydrogels were prepared by the radical polymerization of acrylamide (AAm), and N,N'-methylenebisacrylamide (BIS). By changing the BIS concentration, the elasticity of PAAm hydrogels changed from 1 to 70kPa. For the bio-specific immobilization of ephrinB2, a chimeric protein of ephrinB2 and Fc domain was immobilized onto protein A-conjugated PAAm hydrogels by making use of the bio-specific interaction between the Fc domain and protein A. When hMSC were cultured on the ephrinB2-immobilized PAAm hydrogels with varied elasticity, the morphology of hMSC was of cuboidal shape on the PAAm hydrogels immobilized with ephrinB2 compared with non-conjugated ones, irrespective of the hydrogels elasticity. The bio-specific immobilization of ephrinB2 enhanced the level of Runx2 expression. The expression level was significantly high for the hydrogels of 3.6 and 5.9kPa elasticity with bio-specific immobilization of ephrinB2 compared with other hydrogels with the same elasticity. The hydrogels showed a significantly down-regulated RhoA activity. It is concluded that the Runx2 expression of hMSC is synergistically influenced by the hydrogels elasticity and their immobilized manner of ephrinB2 immobilized. Differentiation fate of mesenchymal stem cells (MSC) is modified by biochemical and biophysical factors, such as elasticity and signal proteins. However, there are few experiments about combinations of them. In this study, to evaluate the synergistic effect of them on cell properties of MSC, we established to design the manner of Eph signal ligand, ephrinB2, immobilized onto polyacrylamide hydrogels with varied elasticity. The gene expression level of an osteogenic maker, Runx2, was enhanced

  19. Co-immobilization of semaphorin3A and nerve growth factor to guide and pattern axons.

    Science.gov (United States)

    McCormick, Aleesha M; Jarmusik, Natalie A; Leipzig, Nic D

    2015-12-01

    distribution. The novelty of this research is that we provide a specific attachment method to immobilize an attractive signal, nerve growth factor, along with an inhibitory cue, semaphorin3A, to a substrate in order to analyze the interplay of these proteins on axon guidance responses. The scientific impact of this manuscript is that we show that dual-cued platforms are necessary in order to finetune and tailor specific axon responses for varying neuronal regenerative purposes. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  20. Properties of immobilized papain by radiation polymerization

    International Nuclear Information System (INIS)

    Kumakura, Minoru; Kaetsu, Isao

    1984-01-01

    Papain was immobilized by the radiation polymerization of various monomers at low temperatures and the effects of the polymer matrix on the enzyme activity and thermal stability of the immobilized enzymes were studied. The activity of the immobilized enzymes prepared from monofunctional (acrylate and methacrylate) monomers was higher than that from bifunctional (bismethacrylate) monomers and that from polyoxyethylene dimethacrylate monomers increased with an increase in the number of oxyethylene units. The thermal stability of the immobilized enzymes prepared from hydrophilic monomers was higher than that from hydrophobic monomers and increased markedly with increasing monomer concentration. (author)

  1. Production of Biodiesel Using Immobilized Lipase and the Characterization of Different Co-Immobilizing Agents and Immobilization Methods

    Directory of Open Access Journals (Sweden)

    Kang Zhao

    2016-08-01

    Full Text Available Lipase from Candida sp. 99–125 is widely employed to catalyzed transesterification and can be used for biodiesel production. In this study, the lipase was immobilized by combined adsorption and entrapment to catalyze biodiesel production from waste cooking oil (WCO via transesterification, and investigating co-immobilizing agents as additives according to the enzyme activity. The addition of the mixed co-immobilizing agents has positive effects on the activities of the immobilized lipase. Three different immobilizing methods were compared by the conversion ratio of biodiesel and structured by Atom Force Microscopy (AFM and Scanning Electron Microscopy (SEM, respectively. It was found that entrapment followed by adsorption was the best method. The effect of the co-immobilizing agent amount, lipase dosage, water content, and reuse ability of the immobilized lipase was investigated. By comparison with previous research, this immobilized lipase showed good reuse ability: the conversion ratio excesses 70% after 10 subsequent reactions, in particular, was better than Novozym435 and TLIM on waste cooking oil for one unit of lipase.

  2. Technetium Immobilization Forms Literature Survey

    Energy Technology Data Exchange (ETDEWEB)

    Westsik, Joseph H.; Cantrell, Kirk J.; Serne, R. Jeffrey; Qafoku, Nikolla

    2014-05-01

    Of the many radionuclides and contaminants in the tank wastes stored at the Hanford site, technetium-99 (99Tc) is one of the most challenging to effectively immobilize in a waste form for ultimate disposal. Within the Hanford Tank Waste Treatment and Immobilization Plant (WTP), the Tc will partition between both the high-level waste (HLW) and low-activity waste (LAW) fractions of the tank waste. The HLW fraction will be converted to a glass waste form in the HLW vitrification facility and the LAW fraction will be converted to another glass waste form in the LAW vitrification facility. In both vitrification facilities, the Tc is incorporated into the glass waste form but a significant fraction of the Tc volatilizes at the high glass-melting temperatures and is captured in the off-gas treatment systems at both facilities. The aqueous off-gas condensate solution containing the volatilized Tc is recycled and is added to the LAW glass melter feed. This recycle process is effective in increasing the loading of Tc in the LAW glass but it also disproportionally increases the sulfur and halides in the LAW melter feed which increases both the amount of LAW glass and either the duration of the LAW vitrification mission or the required supplemental LAW treatment capacity.

  3. Method for selective immobilization of macromolecules on self assembled monolayer surfaces

    Science.gov (United States)

    Laskin, Julia [Richland, WA; Wang, Peng [Billerica, MA

    2011-11-29

    Disclosed is a method for selective chemical binding and immobilization of macromolecules on solid supports in conjunction with self-assembled monolayer (SAM) surfaces. Immobilization involves selective binding of peptides and other macromolecules to SAM surfaces using reactive landing (RL) of mass-selected, gas phase ions. SAM surfaces provide a simple and convenient platform for tailoring chemical properties of a variety of substrates. The invention finds applications in biochemistry ranging from characterization of molecular recognition events at the amino acid level and identification of biologically active motifs in proteins, to development of novel biosensors and substrates for stimulated protein and cell adhesion.

  4. Toward the establishment of standardized in vitro tests for lipid-based formulations, part 3: understanding supersaturation versus precipitation potential during the in vitro digestion of type I, II, IIIA, IIIB and IV lipid-based formulations.

    Science.gov (United States)

    Williams, Hywel D; Sassene, Philip; Kleberg, Karen; Calderone, Marilyn; Igonin, Annabel; Jule, Eduardo; Vertommen, Jan; Blundell, Ross; Benameur, Hassan; Müllertz, Anette; Pouton, Colin W; Porter, Christopher J H

    2013-12-01

    Recent studies have shown that digestion of lipid-based formulations (LBFs) can stimulate both supersaturation and precipitation. The current study has evaluated the drug, formulation and dose-dependence of the supersaturation - precipitation balance for a range of LBFs. Type I, II, IIIA/B LBFs containing medium-chain (MC) or long-chain (LC) lipids, and lipid-free Type IV LBF incorporating different doses of fenofibrate or tolfenamic acid were digested in vitro in a simulated intestinal medium. The degree of supersaturation was assessed through comparison of drug concentrations in aqueous digestion phases (APDIGEST) during LBF digestion and the equilibrium drug solubility in the same phases. Increasing fenofibrate or tolfenamic acid drug loads (i.e., dose) had negligible effects on LC LBF performance during digestion, but promoted drug crystallization (confirmed by XRPD) from MC and Type IV LBF. Drug crystallization was only evident in instances when the calculated maximum supersaturation ratio (SR(M)) was >3. This threshold SR(M) value was remarkably consistent across all LBF and was also consistent with previous studies with danazol. The maximum supersaturation ratio (SR(M)) provides an indication of the supersaturation 'pressure' exerted by formulation digestion and is strongly predictive of the likelihood of drug precipitation in vitro. This may also prove effective in discriminating the in vivo performance of LBFs.

  5. Co-Immobilization of Enzymes and Magnetic Nanoparticles by Metal-Nucleotide Hydrogelnanofibers for Improving Stability and Recycling

    Directory of Open Access Journals (Sweden)

    Chunfang Li

    2017-01-01

    Full Text Available In this paper we report a facile method for preparing co-immobilized enzyme and magnetic nanoparticles (MNPs using metal coordinated hydrogel nanofibers. Candida rugosa lipase (CRL was selected as guest protein. For good aqueous dispersity, low price and other unique properties, citric acid-modified magnetic iron oxide nanoparticles (CA-Fe3O4 NPs have been widely used for immobilizing enzymes. As a result, the relative activity of CA-Fe3O4@Zn/AMP nanofiber-immobilized CRL increased by 8-fold at pH 10.0 and nearly 1-fold in a 50 °C water bath after 30 min, compared to free CRL. Moreover, the immobilized CRL had excellent long-term storage stability (nearly 80% releative activity after storage for 13 days. This work indicated that metal-nucleotide nanofibers could efficiently co-immobilize enzymes and MNPs simultaneously, and improve the stability of biocatalysts.

  6. A preliminary study of continuous milk coagulation using Cynara cardunculus flower extract and calf rennet immobilized on magnetic particles.

    Science.gov (United States)

    Liburdi, Katia; Emiliani Spinelli, Sara; Benucci, Ilaria; Lombardelli, Claudio; Esti, Marco

    2018-01-15

    The aim of this study was to develop a bioreactor design for continuous milk coagulation using a biocatalyst composed of immobilized animal and vegetable rennet on aminated magnetic particles, which has been proven to be an appropriate carrier for enzyme immobilization. Calf and vegetable (Cynara cardunculus) rennets were covalently immobilized on CLEA® magnetic supports and the immobilization procedure was optimized in batch mode, by evaluating protein loading, caseinolytic activity and the coagulation properties of skim milk powder and cow's milk. Subsequently the optimal temperature of immobilized coagulant was defined and a technically-friendly enzyme bioreactor was developed in order to carry out a continuous milk coagulation process with the aim of producing soft cheese. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Lipid-based oral delivery systems for skin deposition of a potential chemopreventive DIM derivative: characterization and evaluation.

    Science.gov (United States)

    Boakye, Cedar H A; Patel, Ketan; Patel, Apurva R; Faria, Henrique A M; Zucolotto, Valtencir; Safe, Stephen; Singh, Mandip

    2016-10-01

    The objective of this study was to explore the oral route as a viable potential for the skin deposition of a novel diindolylmethane derivative (DIM-D) for chemoprevention activity. Various lipid-based oral delivery systems were optimized and compared for enhancing DIM-D's oral bioavailability and skin deposition. Preformulation studies were performed to evaluate the log P and solubility of DIM-D. Microsomal metabolism, P-glycoprotein efflux, and caco-2 monolayer permeability of DIM-D were determined. Comparative evaluation of the oral absorption and skin deposition of DIM-D-loaded various lipid-based formulations was performed in rats. DIM-D showed pH-dependent solubility and a high log P value. It was not a strong substrate of microsomal degradation and P-glycoprotein. SMEDDs comprised of medium chain triglycerides, monoglycerides, and kolliphor-HS15 (36.70 ± 0.42 nm). SNEDDs comprised of long chain triglycerides, cremophor RH40, labrasol, and TPGS (84.00 ± 14.14 nm). Nanostructured lipid carriers (NLC) consisted of compritol, miglyol, and surfactants (116.50 ± 2.12 nm). The blank formulations all showed >70 % cell viability in caco-2 cells. Differential Scanning Calorimetry confirmed the amorphization of DIM-D within the lipid matrices while Atomic Force Microscopy showed particle size distribution similar to the dynamic light scattering data. DIM-D also showed reduced permeation across caco-2 monolayer that was enhanced (p < 0.05) by SNEDDs in comparison to SMEDDs and NLC. Fabsolute for DIM-D SNEDDs, SMEDDs, and NLC was 0.14, 0.04, and 0.007, respectively. SNEDDs caused 53.90, 11.32, and 15.08-fold more skin deposition of DIM-D than the free drug, SMEDDs, and NLC, respectively, at 2 h following oral administration and shows a viable potential for use in skin cancer chemoprevention. Graphical Abstract ᅟ.

  8. Feeding behaviors during home-based treatment of moderate acute malnutrition using corn-soy blends or lipid-based nutrient supplements

    DEFF Research Database (Denmark)

    Iuel-Brockdorff, Ann-Sophie Julie D; Ouedraogo, Albertine; Ritz, Christian

    2017-01-01

    Feeding behaviors have an important impact on children's nutritional status and are essential to consider when implementing nutrition programs. The objective of this study was to explore and compare feeding behaviors related to supplementary feeding with corn-soy blends (CSB) and lipid-based nutr...

  9. Optimization of Adsorptive Immobilization of Alcohol Dehydrogenases

    NARCIS (Netherlands)

    Trivedi, Archana; Heinemann, Matthias; Spiess, Antje C.; Daussmann, Thomas; Büchs, Jochen

    2005-01-01

    In this work, a systematic examination of various parameters of adsorptive immobilization of alcohol dehydrogenases (ADHs) on solid support is performed and the impact of these parameters on immobilization efficiency is studied. Depending on the source of the enzymes, these parameters differently

  10. Plutonium Immobilization Can Loading Equipment Review

    International Nuclear Information System (INIS)

    Kriikku, E.; Ward, C.; Stokes, M.; Randall, B.; Steed, J.; Jones, R.; Hamilton, L.

    1998-05-01

    This report lists the operations required to complete the Can Loading steps on the Pu Immobilization Plant Flow Sheets and evaluates the equipment options to complete each operation. This report recommends the most appropriate equipment to support Plutonium Immobilization Can Loading operations

  11. Drug immobilization of walrus (Odobenus rosmarus)

    Science.gov (United States)

    DeMaster, D.P.; Faro, J.B.; Estes, J.A.; Taggart, James; Zabel, C.

    1981-01-01

    Five out of nine walrus (Odobenus rosmarus) were successfully immobilized at Round Island, Alaska, in May of 1978 by combinations of phencyclidine hydrochloride and acepromazine hydrochloride. A crossbow was an effective delivery technique. Walruses that had recently hauled out were more suitable for immobilization than well-rested animals. Care was taken to prevent walruses from overheating or suffocating.

  12. Immobilization of glucose oxidase to nanostructured films of polystyrene-block-poly(2-vinylpyridine).

    Science.gov (United States)

    Bhakta, Samir A; Benavidez, Tomas E; Garcia, Carlos D

    2014-09-15

    A critical step for the development of biosensors is the immobilization of the biorecognition element to the surface of a substrate. Among other materials that can be used as substrates, block copolymers have the untapped potential to provide significant advantages for the immobilization of proteins. To explore such possibility, this manuscript describes the fabrication and characterization of thin-films of polystyrene-block-poly(2-vinylpyridine) (PS-b-P2VP). These films were then used to investigate the immobilization of glucose oxidase, a model enzyme for the development of biosensors. According to the results presented, the nanoporous films can provide significant increases in surface area of the substrate and the immobilization of larger amounts of active enzyme. The characterization of the substrate-enzyme interface discussed in the manuscript aims to provide critical information about relationship between the surface (material, geometry, and density of pores), the protein structure, and the immobilization conditions (pH, and protein concentration) required to improve the catalytic activity and stability of the enzymes. A maximum normalized activity of 3300±700 U m(-2) was achieved for the nanoporous film of PS-b-P2VP. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Immobilized fluid membranes for gas separation

    Science.gov (United States)

    Liu, Wei; Canfield, Nathan L; Zhang, Jian; Li, Xiaohong Shari; Zhang, Jiguang

    2014-03-18

    Provided herein are immobilized liquid membranes for gas separation, methods of preparing such membranes and uses thereof. In one example, the immobilized membrane includes a porous metallic host matrix and an immobilized liquid fluid (such as a silicone oil) that is immobilized within one or more pores included within the porous metallic host matrix. The immobilized liquid membrane is capable of selective permeation of one type of molecule (such as oxygen) over another type of molecule (such as water). In some examples, the selective membrane is incorporated into a device to supply oxygen from ambient air to the device for electrochemical reactions, and at the same time, to block water penetration and electrolyte loss from the device.

  14. Mapping and identification of HeLa cell proteins separated by immobilized pH-gradient two-dimensional gel electrophoresis and construction of a two-dimensional polyacrylamide gel electrophoresis database

    DEFF Research Database (Denmark)

    Shaw, AC; Rossel Larsen, M; Roepstorff, P

    1999-01-01

    The HeLa cell line, a human adenocarcinoma, is used in many research fields, since it can be infected with a wide range of viruses and intracellular bacteria. Therefore, the mapping of HeLa cell proteins is useful for the investigation of parasite host cell interactions. Because of the recent imp...... these and future data accessible for interlaboratory comparison, we constructed a 2-D PAGE database on the World Wide Web....... the mapping of [35S]methionine/cysteine-labeled HeLa cell proteins with the 2-D PAGE (IPG)-system, using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and N-terminal sequencing for protein identification. To date 21 proteins have been identified and mapped. In order to make...

  15. Immobilization of azurin with retention of its native electrochemical properties at alkylsilane self-assembled monolayer modified indium tin oxide

    International Nuclear Information System (INIS)

    Ashur, Idan; Jones, Anne K.

    2012-01-01

    Highlights: ► Immobilization of azurin at indium tin oxide causes modification of the native redox properties. ► Azurin was immobilized at alkylsilane self-assembled monolayer on indium tin oxide. ► Native, solution redox properties are retained for the immobilized protein on the SAM. ► Technique should be widely applicable to other redox proteins. - Abstract: Indium tin oxide (ITO) is a promising material for developing spectroelectrochemical methods due to its combination of excellent transparency in the visible region and high conductivity over a broad range of potential. However, relatively few examples of immobilization of redox proteins at ITO with retention of the ability to transfer electrons with the underlying material with native characteristics have been reported. In this work, we utilize an alkylsilane functionalized ITO surface as a biocompatible interface for immobilization of the blue copper protein azurin. Adsorption of azurin at ITO as well as ITO coated with self-assembled monolayers of (3-mercaptopropyl)trimethoxysilane (MPTMS) and n-decyltrimethoxysilane (DTMS) was achieved, and immobilized protein probed using protein film electrochemistry. The native redox properties of the protein were perturbed by adsorption directly to ITO or to the MPTMS layer on an ITO surface. However, azurin adsorbed at a DTMS covered ITO surface retained native electrochemical properties (E 1/2 = 122 ± 5 mV vs. Ag/AgCl) and could exchange electrons directly with the underlying ITO layer without need for an intervening chemical mediator. These results open new opportunities for immobilizing functional redox proteins at ITO and developing spectroelectrochemical methods for investigating them.

  16. Novel Nanostructured Solid Materials for Modulating Oral Drug Delivery from Solid-State Lipid-Based Drug Delivery Systems.

    Science.gov (United States)

    Dening, Tahnee J; Rao, Shasha; Thomas, Nicky; Prestidge, Clive A

    2016-01-01

    Lipid-based drug delivery systems (LBDDS) have gained significant attention in recent times, owing to their ability to overcome the challenges limiting the oral delivery of poorly water-soluble drugs. Despite the successful commercialization of several LBDDS products over the years, a large discrepancy exists between the number of poorly water-soluble drugs displaying suboptimal in vivo performances and the application of LBDDS to mitigate their various delivery challenges. Conventional LBDDS, including lipid solutions and suspensions, emulsions, and self-emulsifying formulations, suffer from various drawbacks limiting their widespread use and commercialization. Accordingly, solid-state LBDDS, fabricated by adsorbing LBDDS onto a chemically inert solid carrier material, have attracted substantial interest as a viable means of stabilizing LBDDS whilst eliminating some of the various limitations. This review describes the impact of solid carrier choice on LBDDS performance and highlights the importance of appropriate solid carrier material selection when designing hybrid solid-state LBDDS. Specifically, emphasis is placed on discussing the ability of the specific solid carrier to modulate drug release, control lipase action and lipid digestion, and enhance biopharmaceutical performance above the original liquid-state LBDDS. To encourage the interested reader to consider their solid carrier choice on a higher level, various novel materials with the potential for future use as solid carriers for LBDDS are described. This review is highly significant in guiding future research directions in the solid-state LBDDS field and fostering the translation of these delivery systems to the pharmaceutical marketplace.

  17. Effect of lipid-based dry eye supplements on the tear film in wearers of eye cosmetics.

    Science.gov (United States)

    Wang, Michael T M; Cho, Irene Sung Hee; Jung, Soo Hee; Craig, Jennifer P

    2017-08-01

    To compare the effects on tear film parameters and contamination in cosmetic eyeliner wearers, after single application of two lipid-based dry eye treatments: a lipid-containing lubricant eye drop and a phospholipid liposomal spray. Fifty participants were enrolled in a prospective, randomised, paired-eye, investigator-masked trial. Pencil eyeliner (Body Shop ® Crayon Eye Definer) was applied to the upper eyelid periocular skin of both eyes, anterior to the lash line. Baseline tear film quality was assessed fifteen minutes after eyeliner application. A lubricant drop (Systane ® Balance) was then applied to one eye (randomised), and liposomal spray (Tears Again ® ) to the contralateral eye. Tear film contamination, lipid layer grade, non-invasive tear film break-up time and tear evaporation rate were evaluated fifteen minutes post-treatment and compared to pre-treatment values. Pre-treatment measurements did not differ between eyes assigned to lubricant drop and liposomal spray. Tear film contamination was observed in a greater proportion of eyes following both treatments (both p0.05). Changes in tear film parameters did not differ between treatments (all p>0.05). Both the lipid-containing lubricant eye drop and phospholipid liposomal spray result in clinically apparent tear film contamination in eyeliner cosmetic wearers. Although both treatments effected an increase in lipid layer thickness, neither displayed clinical efficacy in improving tear film stability. Copyright © 2017 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

  18. Assessing cellular toxicities in fibroblasts upon exposure to lipid-based nanoparticles: a high content analysis approach

    Science.gov (United States)

    Solmesky, Leonardo J.; Shuman, Michal; Goldsmith, Meir; Weil, Miguel; Peer, Dan

    2011-12-01

    Lipid-based nanoparticles (LNPs) are widely used for the delivery of drugs and nucleic acids. Although most of them are considered safe, there is confusing evidence in the literature regarding their potential cellular toxicities. Moreover, little is known about the recovery process cells undergo after a cytotoxic insult. We have previously studied the systemic effects of common LNPs with different surface charge (cationic, anionic, neutral) and revealed that positively charged LNPs ((+)LNPs) activate pro-inflammatory cytokines and induce interferon response by acting as an agonist of Toll-like receptor 4 on immune cells. In this study, we focused on the response of human fibroblasts exposed to LNPs and their cellular recovery process. To this end, we used image-based high content analysis (HCA). Using this strategy, we were able to show simultaneously, in several intracellular parameters, that fibroblasts can recover from the cytotoxic effects of (+)LNPs. The use of HCA opens new avenues in understanding cellular response and nanotoxicity and may become a valuable tool for screening safe materials for drug delivery and tissue engineering.

  19. Assessing cellular toxicities in fibroblasts upon exposure to lipid-based nanoparticles: a high content analysis approach

    International Nuclear Information System (INIS)

    Solmesky, Leonardo J; Weil, Miguel; Shuman, Michal; Goldsmith, Meir; Peer, Dan

    2011-01-01

    Lipid-based nanoparticles (LNPs) are widely used for the delivery of drugs and nucleic acids. Although most of them are considered safe, there is confusing evidence in the literature regarding their potential cellular toxicities. Moreover, little is known about the recovery process cells undergo after a cytotoxic insult. We have previously studied the systemic effects of common LNPs with different surface charge (cationic, anionic, neutral) and revealed that positively charged LNPs ((+)LNPs) activate pro-inflammatory cytokines and induce interferon response by acting as an agonist of Toll-like receptor 4 on immune cells. In this study, we focused on the response of human fibroblasts exposed to LNPs and their cellular recovery process. To this end, we used image-based high content analysis (HCA). Using this strategy, we were able to show simultaneously, in several intracellular parameters, that fibroblasts can recover from the cytotoxic effects of (+)LNPs. The use of HCA opens new avenues in understanding cellular response and nanotoxicity and may become a valuable tool for screening safe materials for drug delivery and tissue engineering.

  20. Preventative lipid-based nutrient supplements (LNS) and young child feeding practices: findings from qualitative research in Haiti.

    Science.gov (United States)

    Lesorogol, Carolyn; Jean-Louis, Sherlie; Green, Jamie; Iannotti, Lora

    2015-12-01

    To prevent undernutrition in an urban slum in Haiti, a lipid-based nutrient supplement (LNS) was introduced through a randomised control trial. Food supplementation for young child nutrition has a long history in Haiti, but there is little empirical information regarding the effects of supplementation on young child feeding practices. One of the concerns raised by supplementation is that it may disrupt other positive feeding practices such as breastfeeding and use of other complementary foods, with negative consequences for child nutrition. We conducted 29 in-depth interviews with mother-baby pairs from the three comparison groups: control, 3-month LNS supplementation and 6-month LNS supplementation. Findings from those in the LNS groups indicated high acceptance and satisfaction with LNS and perceptions that it positively affects child health and development. LNS was integrated into and enhanced ongoing complementary feeding practices. The effects of LNS use on duration and perceived quantity of breastfeeding were variable, but generally, breastfeeding was maintained during and after the intervention. Interviews generated insights into beliefs regarding infant and young child feeding practices such as introduction and use of complementary foods, and breastfeeding duration, exclusivity and cessation. Implications for the use of LNS in public health nutrition programmes are discussed. © 2014 John Wiley & Sons Ltd.

  1. Polar Quassinoids in Standardized Eurycoma longifolia Extract Formulated into a Lipid-Based Solid Dispersion to Improve Rat Sperm Count.

    Science.gov (United States)

    Ma, Hai-Qiu; Ebrahimi, Forough; Low, Bin-Seng; Khan, Nurzalina Abdul Karim; Chan, Kit-Lam

    2017-12-01

    Eurycoma longifolia Jack is popularly sought in Southeast Asian countries for traditional remedies to improve sexual performance and fertility. 13α(21)-Epoxyeurycomanone and eurycomanone, two major quassinoids in a root extract (TAF2) were reported to improve rat spermatogenesis and fertility. Unfortunately, these quassinoids possess low bioavailability because of high aqueous solubility and low lipid membrane permeability. Often, other possible barriers may be P-glycoprotein (P-gp) efflux in the gut and presystemic hepatic metabolism. The present study attempted to solve these problems by formulating a lipid-based solid dispersion (TAF2-SD) of optimized mixture of TAF2 and emulsifiers, which was then orally administered to rats prior to sperm count analysis. The TAF2-SD-treated rats showed significantly twofold (p < 0.001) and fourfold (p < 0.001) higher sperm count than did TAF2-treated and vehicle-treated (control) rats, respectively. The study also demonstrated no significant in vitro ileal absorption changes of the quassinoids by P-gp efflux inhibitors and concentration change or secondary metabolite formation upon in vitro incubation with rat liver homogenates, suggesting that P-gp-mediated efflux and presystemic metabolism were not limiting their bioavailability. Further study on orally TAF2-treated rats confirmed that the area under the curve and bioavailability curve of each quassinoid in the absence and presence of ketoconazole were unchanged. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  2. Structural and Chemical Characterization of Silica Spheres before and after Modification by Silanization for Trypsin Immobilization

    Directory of Open Access Journals (Sweden)

    Eduardo F. Barbosa

    2017-01-01

    Full Text Available In the last decades, silica particles of a variety of sizes and shapes have been characterized and chemically modified for several applications, from chromatographic separation to dental supplies. The present study proposes the use of aminopropyl triethoxysilane (APTS silanized silica particles to immobilize the proteolytic enzyme trypsin for the development of a bioreactor. The major advantage of the process is that it enables the polypeptides hydrolysis interruption simply by removing the silica particles from the reaction bottle. Silanized silica surfaces showed significant morphological changes at micro- and nanoscale level. Chemical characterization showed changes in elemental composition, chemical environment, and thermal degradation. Their application as supports for trypsin immobilization showed high immobilization efficiency at reduced immobilization times, combined with more acidic conditions. Indirect immobilization quantification by reversed-phase ultrafast high performance liquid chromatography proved to be a suitable approach due to its high linearity and sensitivity. Immobilized trypsin activities on nonmodified and silanized silica showed promising features (e.g., selective hydrolysis for applications in proteins/peptides primary structure elucidation for proteomics. Silanized silica system produced some preferential targeting peptides, probably due to the hydrophobicity of the nanoenvironment conditioned by silanization.

  3. Improvement of the stability and activity of immobilized glucose oxidase on modified iron oxide magnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Abbasi, Mahboube, E-mail: mahbubeabbasi@yahoo.com; Amiri, Razieh, E-mail: razieh.amiri@gmail.com; Bordbar, Abdol-Kalegh, E-mail: bordbar@chem.ui.ac.ir; Ranjbakhsh, Elnaz, E-mail: e.ranjbakhsh@yahoo.com; Khosropour, Ahmad-Reza, E-mail: khosropour@chem.ui.ac.ir

    2016-02-28

    Graphical abstract: - Highlights: • Modified iron oxide magnetic nanoparticles were synthesized by co-precipitation method and characterized by TEM and XRD. • Covalent attachment of GOX to MIMNs was confirmed by FT-IR technique. • Optimization of the reaction time and initial amount of the GOX were carried out. • Improvement of activity and stability of immobilized GOX have been increased in comparison of free GOX. - Abstract: Immobilized proteins and enzymes are widely investigated in the medical field as well as the food and environmental fields. In this study, glucose oxidase (GOX) was covalently immobilized on the surface of modified iron oxide magnetic nanoparticles (MIMNs) to produce a bioconjugate complex. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to the size, shape and structure characterization of the MIMNs. Binding of GOX to these MIMNs was confirmed by using FT-IR spectroscopy. The stability of the immobilized and free enzyme at different temperature and pH values was investigated by measuring the enzymatic activity. These studies reveal that the enzyme's stability is enhanced by immobilization. Further experiments showed that the storage stability of the enzyme is improved upon binding to the MIMNs. The results of kinetic measurements suggest that the effect of the immobilization process on substrate and product diffusion is small. Such bioconjugates can be considered as a catalytic nanodevice for accelerating the glucose oxidation reaction for biotechnological purposes.

  4. Plutonium Immobilization Can Loading Concepts

    International Nuclear Information System (INIS)

    Kriikku, E.; Ward, C.; Stokes, M.; Randall, B.; Steed, J.; Jones, R.; Hamilton, L.; Rogers, L.; Fiscus, J.; Dyches, G.

    1998-05-01

    The Plutonium Immobilization Facility will encapsulate plutonium in ceramic pucks and seal the pucks inside welded cans. Remote equipment will place these cans in magazines and the magazines in a Defense Waste Processing Facility (DWPF) canister. The DWPF will fill the canister with glass for permanent storage. This report discusses five can loading conceptual designs and the lists the advantages and disadvantages for each concept. This report identifies loading pucks into cans and backfilling cans with helium as the top priority can loading development areas. The can loading welder and cutter are very similar to the existing Savannah River Site (SRS) FB-Line bagless transfer welder and cutter and thus they are a low priority development item

  5. Immobilization of organic liquid wastes

    International Nuclear Information System (INIS)

    Greenhalgh, W.O.

    1985-01-01

    This report describes a portland cement immobilization process for the disposal treatment of radioactive organic liquid wastes which would be generated in a a FFTF fuels reprocessing line. An incineration system already on-hand was determined to be too costly to operate for the 100 to 400 gallons per year organic liquid. Organic test liquids were dispersed into an aqueous phosphate liquid using an emulsifier. A total of 109 gallons of potential and radioactive aqueous immiscible organic liquid wastes from Hanford 300 Area operations were solidified with portland cement and disposed of as solid waste during a 3-month test program with in-drum mixers. Waste packing efficiencies varied from 32 to 40% and included pump oils, mineral spirits, and TBP-NPH type solvents

  6. Uranium Immobilization in Wetland Soils

    Science.gov (United States)

    Jaffe, Peter R.; Koster van Groos, Paul G.; Li, Dien; Chang, Hyun-Shik; Seaman, John C.; Kaplan, Daniel I.; Peacock, Aaron D.; Scheckel, Kirk

    2014-05-01

    stronger for the mesocosms with the higher Fe(II) load. Analysis via XANES showed that a fraction (up to ~1/3) of uranium was reduced to U(IV), for mesocosms operated under low iron loading, indicating that iron cycling in the rhizosphere also results in uranium reduction and immobilization. For mesocosms operating under the higher iron loading, the fraction of uranium immobilized as U(IV) was much lower, indicating that uranium co-precipitation with iron might have been the dominant immobilization process. In parallel to these mesocosm experiments, dialysis samplers have been deployed at the Savannah River National Laboratory near a creek with uranium contamination, to determine dissolved species, including Fe(II) and U(VI) in these wetland soils and their seasonal variability. The results show that there is a strong seasonal variability in dissolved iron and uranium, indicating a strong immobilization during the growing season, which is consistent with the mesocosm experimental results that the rhizosphere iron and uranium cycling are closely linked.

  7. Radioactive seed immobilization techniques for interstitial brachytherapy

    International Nuclear Information System (INIS)

    Yan, K.; Podder, T.; Buzurovic, I.; Hu, Y.; Dicker, A.; Valicenti, R.; Yu, Y.; Messing, E.; Rubens, D.; Sarkar, N.; Ng, W.

    2008-01-01

    In prostate brachytherapy, seeds can detach from their deposited sites and move locally in the pelvis or migrate to distant sites including the pulmonary and cardiac regions. Undesirable consequences of seed migration include inadequate dose coverage of the prostate and tissue irradiation effects at the site of migration. Thus, it is clinically important to develop seed immobilization techniques. We first analyze the possible causes for seed movement, and propose three potential techniques for seed immobilization: (1) surgical glue, (2) laser coagulation and (3) diathermy coagulation. The feasibility of each method is explored. Experiments were carried out using fresh bovine livers to investigate the efficacy of seed immobilization using surgical glue. Results have shown that the surgical glue can effectively immobilize the seeds. Evaluation of the radiation dose distribution revealed that the non-immobilized seed movement would change the planned isodose distribution considerably; while by using surgical glue method to immobilize the seeds, the changes were negligible. Prostate brachytherapy seed immobilization is necessary and three alternative mechanisms are promising for addressing this issue. Experiments for exploring the efficacy of the other two proposed methods are ongoing. Devices compatible with the brachytherapy procedure will be designed in future. (orig.)

  8. Stable functionalization of germanium surface and its application in biomolecules immobilization

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Qi [State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, No.865, Changning Road, Shanghai 200050 (China); University of Chinese Academy of Sciences, No.19A, Yuquan Road, Beijing 100049 (China); Xu, Baojian [State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, No.865, Changning Road, Shanghai 200050 (China); Ye, Lin [Sate Key Laboratory of Functional Materials for Informatics, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, No.865, Changning Road, Shanghai 200050 (China); University of Chinese Academy of Sciences, No.19A, Yuquan Road, Beijing 100049 (China); Tang, Teng; Huang, Shanluo; Du, Xiaowei [State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, No.865, Changning Road, Shanghai 200050 (China); University of Chinese Academy of Sciences, No.19A, Yuquan Road, Beijing 100049 (China); Bian, Xiaojun; Zhang, Jishen [State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, No.865, Changning Road, Shanghai 200050 (China); Di, Zengfeng, E-mail: zfdi@mail.sim.ac.cn [Sate Key Laboratory of Functional Materials for Informatics, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, No.865, Changning Road, Shanghai 200050 (China); Jin, Qinghui [State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, No.865, Changning Road, Shanghai 200050 (China); Zhao, Jianlong, E-mail: jlzhao@mail.sim.ac.cn [State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, No.865, Changning Road, Shanghai 200050 (China)

    2014-10-15

    Highlights: • An effective method to immobilize biomolecules on the functionalized Ge surface. • The surface of Ge was functionalized with 11-Mercaptoundecanoic acid (11-MUA). • Stable and uniform SAMs was obtained on Ge surface after 11-MUA treatment. • The functionalized Ge was employed as substrate for protein immobilization. • Paving the way of Ge for further applications in bioelectronics field. - Abstract: As a typical semiconductor material, germanium (Ge) has the potential to be utilized in microelectronics and bioelectronics. Herein, we present a simple and effective method to immobilize biomolecules on the surface of functionalized Ge. The surface oxide of Ge was removed with the pretreatment of hydrochloric acid and the Cl-terminated Ge reacted with 11-Mercaptoundecanoic acid (11-MUA). The surface of Ge was coated with 11-MUA self-assembled monolayers (SAMs) due to the bonding reaction between the sulfhydryl group of 11-MUA and Cl-terminated Ge. Furthermore, typical biomolecule, a green fluorescent protein was chosen to be immobilized on the surface of the functionalized Ge. Contact angle analysis, atomic force microscopy and X-ray photoelectron spectroscopy were used to study the characteristics including wettability, stability, roughness and component of the functionalized Ge, respectively. Fluorescence microscopy was utilized to indicate the efficiency of protein immobilization on the surface of the functionalized Ge. With these studies, stable and uniform functionalized monolayer was obtained on the surface of Ge after 11-MUA treatment and the functionalized Ge was effectively applied in protein immobilization. Furthermore, this study may pave the way for further applications such as the integration of bioelectronics and biosensors with the attractive semiconductor material-Ge in future work.

  9. Surface cell immobilization within perfluoroalkoxy microchannels

    Energy Technology Data Exchange (ETDEWEB)

    Stojkovič, Gorazd; Krivec, Matic [Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana (Slovenia); Vesel, Alenka [Jožef Stefan Institute, Jamova cesta 39, 1000 Ljubljana (Slovenia); Marinšek, Marjan [Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana (Slovenia); Žnidaršič-Plazl, Polona, E-mail: polona.znidarsic@fkkt.uni-lj.si [Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana (Slovenia)

    2014-11-30

    Graphical abstract: - Highlights: • A very efficient approach for immobilization of cells into microreactors is presented. • It is applicable to various materials, including PFA and cyclic olefin (co)polymers. • It was used to immobilize different prokaryotic and eukaryotic microbes. • Cells were immobilized on the surface in high density and showed good stability. • Mechanisms of APTES interactions with target materials are proposed. - Abstract: Perfluoroalkoxy (PFA) is one of the most promising materials for the fabrication of cheap, solvent resistant and reusable microfluidic chips, which have been recently recognized as effective tools for biocatalytic process development. The application of biocatalysts significantly depends on efficient immobilization of enzymes or cells within the reactor enabling long-term biocatalyst use. Functionalization of PFA microchannels by 3-aminopropyltriethoxysilane (ATPES) and glutaraldehyde was used for rapid preparation of microbioreactors with surface-immobilized cells. X-ray photoelectron spectroscopy and scanning electron microscopy were used to accurately monitor individual treatment steps and to select conditions for cell immobilization. The optimized protocol for Saccharomyces cerevisiae immobilization on PFA microchannel walls comprised ethanol surface pretreatment, 4 h contacting with 10% APTES aqueous solution, 10 min treatment with 1% glutaraldehyde and 20 min contacting with cells in deionized water. The same protocol enabled also immobilization of Escherichia coli, Pseudomonas putida and Bacillus subtilis cells on PFA surface in high densities. Furthermore, the developed procedure has been proved to be very efficient also for surface immobilization of tested cells on other materials that are used for microreactor fabrication, including glass, polystyrene, poly (methyl methacrylate), polycarbonate, and two olefin-based polymers, namely Zeonor{sup ®} and Topas{sup ®}.

  10. Immobilization of biomolecules to plasma polymerized pentafluorophenyl methacrylate.

    Science.gov (United States)

    Duque, Luis; Menges, Bernhard; Borros, Salvador; Förch, Renate

    2010-10-11

    Thin films of plasma polymerized pentafluorophenyl methacrylate (pp-PFM) offer highly reactive ester groups throughout the structure of the film that allow for subsequent reactions with different aminated reagents and biological molecules. The present paper follows on from previous work on the plasma deposition of pentafluorophenyl methacrylate (PFM) for optimum functional group retention (Francesch, L.; Borros, S.; Knoll, W.; Foerch, R. Langmuir 2007, 23, 3927) and reactivity in aqueous solution (Duque, L.; Queralto, N.; Francesch, L.; Bumbu, G. G.; Borros, S.; Berger, R.; Förch, R. Plasma Process. Polym. 2010, accepted for publication) to investigate the binding of a biologically active peptide known to induce cellular adhesion (IKVAV) and of biochemically active proteins such as BSA and fibrinogen. Analyses of the films and of the immobilization of the biomolecules were carried out using infrared reflection absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The attachment of the biomolecules on pulsed plasma polymerized pentafluorophenyl methacrylate was monitored using surface plasmon resonance spectroscopy (SPR). SPR analysis confirmed the presence of immobilized biomolecules on the plasma polymer and was used to determine the mass coverage of the peptide and proteins adsorbed onto the films. The combined analysis of the surfaces suggests the covalent binding of the peptide and proteins to the surface of the pp-PFM.

  11. Immobilization of cellulase using porous polymer matrix

    International Nuclear Information System (INIS)

    Kumakura, M.; Kaetsu, I.

    1984-01-01

    A new method is discussed for the immobilization of cellulase using porous polymer matrices, which were obtained by radiation polymerization of hydrophilic monomers. In this method, the immobilized enzyme matrix was prepared by enzyme absorbtion in the porous polymer matrix and drying treatment. The enzyme activity of the immobilized enzyme matrix varied with monomer concentration, cooling rate of the monomer solution, and hydrophilicity of the polymer matrix, takinn the change of the nature of the porous structure in the polymer matrix. The leakage of the enzymes from the polymer matrix was not observed in the repeated batch enzyme reactions

  12. Separations using biological carriers immobilized in porous polymeric and sol-gel template synthesized nanotubular membranes

    Science.gov (United States)

    Lakshmi, Brinda B.

    1998-12-01

    The overall goal of the dissertation was to use immobilized biological carriers in membranes to separate compounds as challenging as enantiomers. The membranes were prepared by a process called 'template synthesis'. Template synthesis has been used to synthesize semiconductor nanostructures and also membranes which do the enantioseparation by a process called facilitated transport. The immobilized proteins act as carriers facilitating the transport of the substrate molecules through the membrane. The apoenzymes are enzymes devoid of cofactor. Apoenzymes will possess the molecular recognition site for the substrate but will not catalyze the reaction. Apoenzymes immobilized in the pores of porous polycarbonate membrane was used as a carrier. The ends of the pores were closed with porous polypyrrole. Compounds as interesting as enantiomers were separated with these membranes. Template synthesis has been extended to the synthesis of many important semiconductor oxide naostructures like TiO2, SiO2, ZnO, Co3O4 and MnO2. These structures were made by dipping the alumina template membrane in the sol and heating. Ti0 2 tubules and fibers were obtained by this method. The fibers were used to study photocatalysis reaction of organic compounds in sunlight. Proteins were immobilized within the inner surface of the tubules using Sn chemistry. Bovine serum albumn (BSA) immobilized within the different diameter tubules showed varying degree of facilitation with phenylalanine. The membranes also show interesting switching of selectivity from L to D depending on the tube size and feed concentration.

  13. Willingness to pay for lipid-based nutrient supplements for young children in four urban sites of Ethiopia.

    Science.gov (United States)

    Segrè, Joel; Winnard, Kim; Abrha, Teweldebrhan Hailu; Abebe, Yewelsew; Shilane, David; Lapping, Karin

    2015-12-01

    Malnutrition in children under 5 years of age is pervasive in Ethiopia across all wealth quintiles. The objective of this study was to determine the willingness to pay (WTP) for a week's supply of Nutributter® (a lipid-based nutrient supplement, or LNS) through typical urban Ethiopian retail channels. In February, 2012, 128 respondents from 108 households with 6-24-month-old children had the opportunity to sample Nutributter® for 2 days in their homes as a complementary food. Respondents were asked directly and indirectly what they were willing to pay for the product, and then participated in market simulation where they could demonstrate their WTP through an exchange of real money for real product. Nearly all (96%) of the respondents had a positive WTP, and 25% were willing to pay the equivalent of at least $1.05, which we calculated as the likely minimum, unsubsidised Ethiopian retail price of Nutributter® for 1 week for one child. Respondents willing to pay at least $1.05 included urban men and women with children 6-24 months old from low-, middle- and high-wealth groups from four study sites across three cities. Additionally, we estimated the initial annual market size for Nutributter® in the cities where the study took place to be around $500 000. The study has important implications for retail distribution of LNS in Ethiopia, showing who the most likely customers could be, and also suggesting why the initial market may be too small to be of interest to food manufacturers seeking profit maximisation. © 2012 Blackwell Publishing Ltd.

  14. Drug Solubility in Fatty Acids as a Formulation Design Approach for Lipid-Based Formulations: A Technical Note.

    Science.gov (United States)

    Lee, Yung-Chi; Dalton, Chad; Regler, Brian; Harris, David

    2018-06-06

    Lipid-based drug delivery systems have been intensively investigated as a means of delivering poorly water-soluble drugs. Upon ingestion, the lipases in the gastrointestinal tract digest lipid ingredients, mainly triglycerides, within the formulation into monoglycerides and fatty acids. While numerous studies have addressed the solubility of drugs in triglycerides, comparatively few publications have addressed the solubility of drugs in fatty acids, which are the end product of digestion and responsible for the solubility of drug within mixed micelles. The objective of this investigation was to explore the solubility of a poorly water-soluble drug in fatty acids and raise the awareness of the importance of drug solubility in fatty acids. The model API (active pharmaceutical ingredient), a weak acid, is considered a BCS II compound with an aqueous solubility of 0.02 μg/mL and predicted partition coefficient >7. The solubility of API ranged from 120 mg/mL to over 1 g/mL in fatty acids with chain lengths across the range C18 to C6. Hydrogen bonding was found to be the main driver of the solubilization of API in fatty acids. The solubility of API was significantly reduced by water uptake in caprylic acid but not in oleic acid. This report demonstrates that solubility data generated in fatty acids can provide an indication of the solubility of the drug after lipid digestion. This report also highlights the importance of measuring the solubility of drugs in fatty acids in the course of lipid formulation development.

  15. Preparation and characterization of immobilized lipase on magnetic hydrophobic microspheres

    DEFF Research Database (Denmark)

    Guo, Zheng; Bai, Shu; Sun, Yan

    2003-01-01

    H for the immobilized CCL were determined. Activity amelioration of the immobilized CCL for the hydrolysis of olive oil was observed, indicating an interfacial activation of the enzyme after immobilization. Moreover, the immobilized CCL showed enhanced thermal stability and good durability in the repeated use after...

  16. Production and immobilization of alpha amylase using biotechnology techniques for use in biological and medical applications

    International Nuclear Information System (INIS)

    Mobasher, E.E.F.

    2009-01-01

    The immobilized enzymes on polymeric supports are prepared for purpose of repeated use and the possibilities of continuous reaction system. One of the most important properties is the stability of proteins when they are used in some medical and industrial applications. The immobilization of the enzymes improves this property as well as many other properties.In this study, alpha amylase was purified and immobilized onto two different polymers. α- amylase was used in this study for its biological and industrial applications. It is used in paper textile, pharmaceutical applications, food, and detergent industries. α- amylase was found in plants, animals, and microorganisms. Purification of α-amylase from microorganisms is the main source of α-amylase because it was excreted from many bacteria and fungi. In this study, α-amylase was purified from Aspergillus niger. Fractional precipitation of the α- amylase produced by A. niger with 80% ammonium sulphate saturation. The crude enzyme was applied on column chromatography packed with Sephadex G 100 for purification. The active eluents containing partially purified enzyme were collected for further investigation. The specific activity of α-amylase was (34.9 U/mg) which was corresponding to 2.09 fold purification for the tested organism. The purified α-amylase was immobilized by entrapment method into two types of polymers. One of them was natural consist of chitosan and alginate. The other polymer was synthetic consist of N- isopropyl acrylamide and alginate. The temperature optimum and thermal inactivation showed a severe loss in the activity of the free enzymes, while the temperature profile of the immobilized enzymes was much broader at higher temperatures demonstrating the effectiveness of the polymer protecting the enzymes. Also, the immobilized enzymes (natural polymer and synthetic polymer) showed higher thermal stability. Optimum ph and stability showed that immobilization of enzymes resulted in more

  17. Catalytic Activity and Photophysical Properties of Biomolecules Immobilized on Mesoporous Silica

    DEFF Research Database (Denmark)

    Ikemoto, Hideki

    Mesoporous silicas, based on Santa Barbara Amorphous-15 (SBA-15), with different morphology, structure, pore size and functional groups have been synthesized. Two metalloenzymes and a photosynthetic membrane protein were immobilized on or confined in the pores of the mesoporous silicas to prepare...

  18. Immobilized unfolded cytochrome c acts as a catalyst for dioxygen reduction.

    Science.gov (United States)

    Tavagnacco, Claudio; Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia; Borsari, Marco

    2011-10-21

    Unfolding turns immobilized cytochrome c into a His-His ligated form endowed with catalytic activity towards O(2), which is absent in the native protein. Dioxygen could be used by naturally occurring unfolded cytochrome c as a substrate for the production of partially reduced oxygen species (PROS) contributing to the cell oxidative stress.

  19. Immobilization of indigenous holocellulase on iron oxide (Fe2O3) nanoparticles enhanced hydrolysis of alkali pretreated paddy straw.

    Science.gov (United States)

    Kumar, Ajay; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata

    2017-03-01

    The holocellulase from Aspergillus niger SH3 was characterized and found to contain 125 proteins including cellulases (26), hemicellulases (21), chitinases (10), esterases (6), amylases (4) and hypothetical protein (32). The crude enzyme was immobilized on five different nanoparticles (NPs) via physical adsorption and covalent coupling methods. The enzyme-nanoparticle complexes (ENC) were screened for protein binding, enzymatic activities and immobilization efficiency. Magnetic enzyme-nanoparticle complexes (MENC) showed higher immobilization efficiency (60-80%) for most of the enzymes. MENC also showed better catalytic efficiencies in term of higher V max and lower K m than free enzyme. Saccharification yields from alkali treated paddy straw were higher (375.39mg/gds) for covalently immobilized MENC than free enzyme (339.99mg/gds). The immobilized enzyme was used for two cycles of saccharification with 55% enzyme recovery. Hence, this study for the first time demonstrated the immobilization of indigenous enzyme and its utilization for saccharification of paddy straw. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Antimicrobial activity of immobilized lactoferrin and lactoferricin.

    Science.gov (United States)

    Chen, Renxun; Cole, Nerida; Dutta, Debarun; Kumar, Naresh; Willcox, Mark D P

    2017-11-01

    Lactoferrin and lactoferricin were immobilized on glass surfaces via two linkers, 4-azidobenzoic acid (ABA) or 4-fluoro-3-nitrophenyl azide (FNA). The resulting surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and contact angle measurements. The antimicrobial activity of the surfaces was determined using Pseudomonas aeruginosa and Staphylococcus aureus strains by fluorescence microscopy. Lactoferrin and lactoferricin immobilization was confirmed by XPS showing significant increases (p lactoferricin immobilized on glass significantly (p lactoferricin were successfully immobilized on glass surfaces and showed promising antimicrobial activity against pathogenic bacteria. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2612-2617, 2017. © 2016 Wiley Periodicals, Inc.

  1. Immobilization of Rocky Flats Graphite Fines Residue

    International Nuclear Information System (INIS)

    Rudisill, T.S.

    1999-01-01

    The development of the immobilization process for graphite fines has proceeded through a series of experimental programs. The experimental procedures and results from each series of experiments are discussed in this report

  2. Immobilization and characterization of inulinase from Ulocladium ...

    Indian Academy of Sciences (India)

    2014-10-20

    Oct 20, 2014 ... The enzyme had optimum pH at 5.6 for free and immobilized U. atrum inulinase on polyester ... ceutical industry because of their beneficial effects in ..... Hanover LWJ 1993 Manufacturing, composing and applications of.

  3. Plutonium Immobilization Bagless Transfer Can Size Evaluation

    International Nuclear Information System (INIS)

    Kriikku, E.; Stokes, M.; Rogers, L.; Ward, C.

    1998-02-01

    This report identifies and documents the most appropriate bagless transfer can size to support Plutonium Immobilization Can Loading operations. Also, this report considers can diameter, can wall thickness, and can length

  4. Modeling intrinsic kinetics in immobilized photocatalytic microreactors

    NARCIS (Netherlands)

    Visan, Aura; Rafieian Boroujeni, Damon; Ogieglo, Wojciech; Lammertink, Rob G.H.

    2014-01-01

    The article presents a simple model for immobilized photocatalytic microreactors following a first order reaction rate with either light independency or light dependency described by photon absorption carrier generation semiconductor physics. Experimental data obtained for various residence times,

  5. A simplified technique for nasoendotracheal tube immobilization.

    OpenAIRE

    Berardo, N.; Leban, S. G.; Williams, F. A.

    1989-01-01

    A simplified technique for immobilization of a nasoendotracheal tube is described in which a wide strap of open cell, hypoallergenic, foam-backed fabric is secured to the patient's head with a Velcro fastener.

  6. [Effects of a lipid-based nutrient supplement on hemoglobin levels and anthropometric indicators in children from five districts in Huánuco Peru].

    Science.gov (United States)

    Vargas-Vásquez, Alejandro; Bado, Ricardo; Alcázar, Lorena; Aquino, Oscar; Rodríguez, Amelia; Novalbos, José Pedro

    2015-01-01

    Objectives . To determine the effect of consumption of a lipid-based nutrient supplement (LNS) on hemoglobin levels, anemia and anthropometric indicators in children aged six to eleven months old in five districts in the province of Ambo in the region of Huanuco, Peru. A pre-experimental, pre-post study was performed. The study population included children aged six to eleven months old in 19 health facilities in five districts with very high vulnerability to chronic malnutrition in the province of Ambo, Huanuco. Data from 147 children who received the lipid-based nutrient supplement (LNS) from six to eleven months old were obtained. The mean hemoglobin significantly increased by 0.67 g/dL (panemia dropped by 27 percentage points (panemia in children under twelve months, which might constitute an effective alternative to prevent and control childhood anemia.

  7. Immobilization technology for krypton in amorphous zeolite

    International Nuclear Information System (INIS)

    Takusagawa, Atsushi; Ishiyama, Keiichi

    1989-01-01

    Radioactive krypton recovered from the offgas of a reprocessing plant requires long-term storage on the order of 100 years. Immobilization technology for krypton into amorphous zeolite 5A is considered one of the best methods for long-term storage. In this report, conditions for immobilization treatment and stability of amorphous zeolite 5A loaded krypton against heat, radiation and water are discussed, and a treatment system using this technology is described. (author)

  8. Immobilization Technologies in Probiotic Food Production

    Directory of Open Access Journals (Sweden)

    Gregoria Mitropoulou

    2013-01-01

    Full Text Available Various supports and immobilization/encapsulation techniques have been proposed and tested for application in functional food production. In the present review, the use of probiotic microorganisms for the production of novel foods is discussed, while the benefits and criteria of using probiotic cultures are analyzed. Subsequently, immobilization/encapsulation applications in the food industry aiming at the prolongation of cell viability are described together with an evaluation of their potential future impact, which is also highlighted and assessed.

  9. Tentacle carrier for immobilization of potato phenoloxidase and its application for halogenophenols removal from aqueous solutions

    Energy Technology Data Exchange (ETDEWEB)

    Loncar, Nikola, E-mail: nloncar@chem.bg.ac.rs [Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade (Serbia); Vujcic, Zoran, E-mail: zvujcic@chem.bg.ac.rs [Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade (Serbia)

    2011-11-30

    Highlights: Black-Right-Pointing-Pointer We synthesized novel immobilization carrier from reused DEAE-cellulose. Black-Right-Pointing-Pointer We used it for immobilization of PPO through coordinative bonding with copper ions. Black-Right-Pointing-Pointer Immobilized PPO showed better characteristics than soluble PPO. Black-Right-Pointing-Pointer TC-PPO removed over 90% of halogenophenols at concentration of 100 mg/L. - Abstract: Halogenated compounds represent one of the most dangerous environmental pollutants, due to their widespread usage as biocides, fungicides, disinfectants, solvent and other industrial chemicals. Immobilization of a protein through coordinate bonds formed with divalent metal ions is becoming an attractive method due to its reversible nature, since the protein may be easily removed from the support matrix through interruption of the protein-metal bond hence giving inherently cleaner and cheaper technology for wastewater treatment. We have synthesized novel 'tentacle' carrier (TC) and used it for immobilization of partially purified potato polyphenol oxidase (PPO). The obtained biocatalyst TC-PPO showed pH optimum at 7.0-8.0 and temperature optimum at 25 Degree-Sign C. Immobilized PPO shows almost 100% of activity at 0 Degree-Sign C. TC-PPO was more resistant to the denaturation induced by sodium dodecyl sulphate (SDS) detergent as compared to its soluble counterpart and was even slightly activated at SDS concentration of 1%. TC-PPO was tested in the batch reactor for 4-chlorophenol and 4-bromophenol removal. More than 90% removal was achieved for both halogenophenols at concentration of 100 mg/L from aqueous solution. For both halogenophenols TC-PPO works with over 90% removal during first three cycles which decrease to 60% removal efficiency after six cycles each of 8 h duration.

  10. The Use of Quasi-Isothermal Modulated Temperature Differential Scanning Calorimetry for the Characterization of Slow Crystallization Processes in Lipid-Based Solid Self-Emulsifying Systems

    OpenAIRE

    Otun, Sarah O.; Meehan, Elizabeth; Qi, Sheng; Craig, Duncan Q. M.

    2014-01-01

    Purpose Slow or incomplete crystallization may be a significant manufacturing issue for solid lipid-based dosage forms, yet little information is available on this phenomenon. In this investigation we suggest a novel means by which slow solidification may be monitored in Gelucire 44/14 using quasi-isothermal modulated temperature DSC (QiMTDSC). Methods Conventional linear heating and cooling DSC methods were employed, along with hot stage microscopy (HSM), for basic thermal profiling of Geluc...

  11. Ceramification: A plutonium immobilization process

    Energy Technology Data Exchange (ETDEWEB)

    Rask, W.C. [Dept. of Energy, Golden, CO (United States); Phillips, A.G. [Rocky Flats Environmental Technology Site, Golden, CO (United States)

    1996-05-01

    This paper describes a low temperature technique for stabilizing and immobilizing actinide compounds using a combination process/storage vessel of stainless steel, in which measured amounts of actinide nitrate solutions and actinide oxides (and/or residues) are systematically treated to yield a solid article. The chemical ceramic process is based on a coating technology that produces rare earth oxide coatings for defense applications involving plutonium. The final product of this application is a solid, coherent actinide oxide with process-generated encapsulation that has long-term environmental stability. Actinide compounds can be stabilized as pure materials for ease of re-use or as intimate mixtures with additives such as rare earth oxides to increase their degree of proliferation resistance. Starting materials for the process can include nitrate solutions, powders, aggregates, sludges, incinerator ashes, and others. Agents such as cerium oxide or zirconium oxide may be added as powders or precursors to enhance the properties of the resulting solid product. Additives may be included to produce a final product suitable for use in nuclear fuel pellet production. The process is simple and reduces the time and expense for stabilizing plutonium compounds. It requires a very low equipment expenditure and can be readily implemented into existing gloveboxes. The process is easily conducted with less associated risk than proposed alternative technologies.

  12. Biotechnological production of vanillin using immobilized enzymes.

    Science.gov (United States)

    Furuya, Toshiki; Kuroiwa, Mari; Kino, Kuniki

    2017-02-10

    Vanillin is an important and popular plant flavor, but the amount of this compound available from plant sources is very limited. Biotechnological methods have high potential for vanillin production as an alternative to extraction from plant sources. Here, we report a new approach using immobilized enzymes for the production of vanillin. The recently discovered oxygenase Cso2 has coenzyme-independent catalytic activity for the conversion of isoeugenol and 4-vinylguaiacol to vanillin. Immobilization of Cso2 on Sepabeads EC-EA anion-exchange carrier conferred enhanced operational stability enabling repetitive use. This immobilized Cso2 catalyst allowed 6.8mg yield of vanillin from isoeugenol through ten reaction cycles at a 1mL scale. The coenzyme-independent decarboxylase Fdc, which has catalytic activity for the conversion of ferulic acid to 4-vinylguaiacol, was also immobilized on Sepabeads EC-EA. We demonstrated that the immobilized Fdc and Cso2 enabled the cascade synthesis of vanillin from ferulic acid via 4-vinylguaiacol with repetitive use of the catalysts. This study is the first example of biotechnological production of vanillin using immobilized enzymes, a process that provides new possibilities for vanillin production. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Radiation technology for immobilization of bioactive materials

    International Nuclear Information System (INIS)

    1988-12-01

    Within the framework of the Agency's coordinated research programme on ''Application of Radiation Technology in Immobilization of Bioactive Materials'', the third and final research coordination meeting was held at Beijing University, Beijing, People's Republic of China, 15-18 June 1987. The present publication compiles all presentations made at the meeting. Fundamental processes for the immobilization of enzymes, antibodies, cells and drugs were developed and established using gamma radiation, electron beams and plasma discharge. Applications of various biofunctional components, immobilized by radiation techniques in different processes, were studied. A range of backbone polymers has been examined together with various monomers. Coupling procedures have been developed which are relevant to our particular requirements. Enzymes of various types and characteristics have been immobilized with considerable efficiency. The immobilized biocatalysts have been shown to possess significant activity and retention of activity on storage. There appears to be a high degree of specificity associated with the properties of the immobilised biocatalysts, their activity and the ease of their preparation. Novel additives which lower the total radiation dose in grafting have been discovered and their value in immobilization processes assessed. Potential applications include: medical (diagnostic, therapeutic), and industrial processes (fermentation, bioseparation, etc.). Refs, figs and tabs

  14. Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors

    Directory of Open Access Journals (Sweden)

    Hui Jian

    2016-07-01

    Full Text Available Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570.

  15. Optimization of antibody immobilization for on-line or off-line immunoaffinity chromatography

    DEFF Research Database (Denmark)

    Beyer, Natascha Helena; Schou, Christian; Højrup, Peter

    2009-01-01

    -POROS. Protein G-based matrices are very stable showing essentially no decline in performance after 50 application-wash-elution-reequilibration cycles and being easily prepared within 2-3 h of working time with a typical antibody coupling yield of above 80%. In off-line applications where constant flow....... A systematic study was conducted to determine the most versatile antibody immobilization method for use in on-line and off-line IA chromatography applications using commonly accessible immobilization methods. Four chemistries were examined using polyclonal and monoclonal antibodies and antibody fragments. We...

  16. Immobilization of myoglobin in sodium alginate composite membranes

    Directory of Open Access Journals (Sweden)

    Katia Cecília de Souza Figueiredo

    2015-06-01

    Full Text Available AbstractThe immobilization of myoglobin in sodium alginate films was investigated with the aim of evaluating the protein stability in an ionic polymeric matrix. Myoglobin was chosen due to the resemblance to each hemoglobin tetramer. Sodium alginate, being a natural polysaccharide, was selected as the polymeric matrix because of its chemical structure and film-forming ability. To improve the mechanical resistance of sodium alginate films, the polymer was deposited over the surface of a cellulose acetate support by means of ultrafiltration. The ionic crosslink of sodium alginate was investigated by calcium ions. Composite membrane characterization comprised water swelling tests, water flux, SEM images and UV-visible spectroscopy. The electrostatic interaction between the protein and the polysaccharide did not damage the UV-visible pattern of native myoglobin. A good affinity between sodium alginate and cellulose acetate was observed. The top layer of the dense composite membrane successfully immobilized Myoglobin, retaining the native UV-visible pattern for two months.

  17. Development of Cellulose Nano fibre (CNF) Derived From Kenaf Bast Fibre and Its Potential in Enzyme Immobilization Support

    International Nuclear Information System (INIS)

    Safwan Sulaiman; Mohd Noriznan Mokhtar; Mohd Nazli Naim; Azhari Samsu Baharuddin

    2016-01-01

    This research mainly focuses on developing a natural cellulose nano fibre (CNF) from kenaf bast fibre and its potential for enzyme immobilization support. CNF was isolated by using a combination between chemical and mechanical treatments such as alkaline process and high-intensity ultrasonication process to increase the efficiency of hemicelluloses and lignin removal, and to reduce its size into nano-order. The morphological study was carried out by using scanning electron microscope (SEM), indicating most of CNF diameter in range of 50-90 nm was obtained. The result of chemical analysis shows that cellulose content of raw bast fibre, bleached pulp fibre and CNF are 66.4 %, 83.7 % and 90.0 %, respectively. By decreasing the size of cellulose fibre, it increases the number of (O-H) group on the surface that plays as important role in enzyme immobilization. Covalent immobilization of cyclodextrin glucanotransferase (CGTase) onto CNF support resulted in about 95.0 % of protein loading with 69.48 % of enzyme activity, indicating high immobilization yield of enzyme. The enzymatic reaction of immobilized CGTase was able to produce more than 40 % yield of α-CD. Reusability profile of immobilized CGTase resulted in more than 60 % of retained activity up to 7 cycles. Therefore, the CNF is highly potential to be applied as enzyme immobilization support. (author)

  18. Outcomes associated with conventional versus lipid-based formulations of amphotericin B in propensity-matched groups

    Directory of Open Access Journals (Sweden)

    Campbell RS

    2013-10-01

    Full Text Available Rebecca S Campbell,1 Paresh Chaudhari,2 Harlen D Hays,1 Robert J Taylor,1 Brian H Nathanson,3 Samuel A Bozzette,1 David Horn4 1Cerner Research, Culver City, CA, USA; 2Astellas Scientific and Medical Affairs, Inc., Northbrook, IL, USA; 3OptiStatim, LLC, Longmeadow, MA, USA; 4David Horn LLC, Doylestown, PA, USA Background: Lipid-based formulations of amphotericin B (LF-AMB are indicated for treatment of invasive fungal infections in patients intolerant to conventional amphotericin B (CAB or with refractory infections. Physicians still may choose to administer CAB to such patients. We described the use of CAB and LF-AMB in this population and quantified differences in post-amphotericin B length of stay (LOS among survivors and hospital mortality in matched patients. Methods: Data were extracted from Health Facts (Cerner Corporation, Kansas City, MO, USA for a retrospective cohort analysis. Inpatients aged ≥18 years with evidence of fungal infection and with orders for LF-AMB or CAB on  ≥2 days from January 2001 to June 2010 were identified. Patients were required to have renal insufficiency or other relative contraindications to use of CAB, exposure to nephrotoxic agents, or evidence of a CAB-refractory infection. Multilevel (hierarchical mixed-effects logistic regression was used to determine factors associated with initial exposure to LF-AMB versus CAB. Multivariate adjustment of outcomes was done using propensity score matching. Results: 655 patients were identified: 322 patients initiated therapy with CAB and 333 initiated treatment with LF-AMB. Compared to those initiating CAB, patients initiating LF-AMB had greater acuity and underlying disease severity. In unadjusted analyses, hospital mortality was significantly higher in the LF-AMB group (32.2% versus 23.7%; P = 0.02. After propensity score matching and covariate adjustment, mortality equalized and observed differences in LOS after amphotericin B initiation decreased. Conclusion

  19. preparation and properties of immobilized naringinase in polyvinyl alcohol copolymer carrier for debittering of citrus juices

    International Nuclear Information System (INIS)

    ElBatal, A.L.; Swailam, H.M.H.

    2003-01-01

    Naringin a bitter compound in citrus can be converted to a nonbiter fom by enzyme hydrolysis. Naringinase from gamma irradiated enhanced Aspergillus nigro-AH3. γ 20 isolate was partoally purified by various methods and the specificacitivty of 6.1 units per mg protein was obtained. The enzyme was immobilized by entrapment in polyvinyl alcohol / polyacrylamide (PVA/PAAm) copolymer hydrogel carrier produced by gamma radiation polymerization. The activity of the immobilized enzyme was elevated with increasing PVA content in the copolymer carrier to reach its maximum value at PVA/PAAm composition ratio of (60.40, v/v%). The yield of immobilization was highest 95% and yield of activity was 91.1%, when 1 mg naringinase enzyme was immobilized in 1 ml of hydrogel matrix. Some enzymatic and compared with those of the solube free enzyme. The optimal pH of the immobilized enzyme was shifted 0.5 pH units to the alkaline side of that of the soluble free enzyme. Also the optimum temperature was shifted from 50 C degree to 60 c degree and the activation energy of reaction (EA) was markedly decreased from 21.52 to 10.74kcal/mol by immobilization. The influence of the diffusion is reflected in the kinetic parameters Km and Vmax. The application of kinetic parameter of the immobilized enzyme optimized with pure naringin solution and bitter orange juice resuled in about 83.2% and 72.0% debittering after 8 h and 12 h incubation during batcheise operation, respectively. The process has operational versatility from the broad pH and temperature optima and affords highest efficiency and stability during application in debittering of bitter orange juice because it is recycled consecutively 14 times before retaining only about 50 % efficiency. These data can be used for future improvements in debittering of citrus fruits products

  20. Immobilization of trypsin on miniature incandescent bulbs for infrared-assisted proteolysis

    Energy Technology Data Exchange (ETDEWEB)

    Ge, Huimin; Bao, Huimin; Zhang, Luyan; Chen, Gang, E-mail: gangchen@fudan.edu.cn

    2014-10-03

    Highlights: • Trypsin was immobilized on miniature incandescent bulbs via chitosan coating. • The bulbs acted as enzymatic reactors and the generators of infrared radiation. • The bulb bioreactors were successfully employed in infrared-assisted proteolysis. • The proteolysis could accomplish within 5 min with high sequence coverages. - Abstract: A novel efficient proteolysis approach was developed based on trypsin-immobilized miniature incandescent bulbs and infrared (IR) radiation. Trypsin was covalently immobilized in the chitosan coating on the outer surface of miniature incandescent bulbs with the aid of glutaraldehyde. When an illuminated enzyme-immobilized bulb was immersed in protein solution, the emitted IR radiation could trigger and accelerate heterogeneous protein digestion. The feasibility and performance of the novel proteolysis approach were demonstrated by the digestion of hemoglobin (HEM), cytochrome c (Cyt-c), lysozyme (LYS), and ovalbumin (OVA) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI-TOF-MS with the sequence coverages of 91%, 77%, 80%, and 52% for HEM, Cyt-c, LYS, and OVA (200 ng μL{sup −1} each), respectively. The suitability of the prepared bulb bioreactors to complex proteins was demonstrated by digesting human serum.

  1. Fabrication of Aligned Carbon Nanotube/Polycaprolactone/Gelatin Nanofibrous Matrices for Schwann Cell Immobilization

    Directory of Open Access Journals (Sweden)

    Shiao-Wen Tsai

    2014-01-01

    Full Text Available In this study, we utilized a mandrel rotating collector consisting of two parallel, electrically conductive pieces of tape to fabricate aligned electrospun polycaprolactone/gelatin (PG and carbon nanotube/polycaprolactone/gelatin (PGC nanofibrous matrices. Furthermore, we examined the biological performance of the PGC nanofibrous and film matrices using an in vitro culture of RT4-D6P2T rat Schwann cells. Using cell adhesion tests, we found that carbon nanotube inhibited Schwann cell attachment on PGC nanofibrous and film matrices. However, the proliferation rates of Schwann cells were higher when they were immobilized on PGC nanofibrous matrices compared to PGC film matrices. Using western blot analysis, we found that NRG1 and P0 protein expression levels were higher for cells immobilized on PGC nanofibrous matrices compared to PG nanofibrous matrices. However, the carbon nanotube inhibited NRG1 and P0 protein expression in cells immobilized on PGC film matrices. Moreover, the NRG1 and P0 protein expression levels were higher for cells immobilized on PGC nanofibrous matrices compared to PGC film matrices. We found that the matrix topography and composition influenced Schwann cell behavior.

  2. Electron paramagnetic resonance spin label titration: a novel method to investigate random and site-specific immobilization of enzymes onto polymeric membranes with different properties

    International Nuclear Information System (INIS)

    Butterfield, D. Allan; Colvin, Joshua; Liu Jiangling; Wang Jianquan; Bachas, Leonidas; Bhattacharrya, Dibakar

    2002-01-01

    The immobilization of biological molecules onto polymeric membranes to produce biofunctional membranes is used for selective catalysis, separation, analysis, and artificial organs. Normally, random immobilization of enzymes onto polymeric membranes leads to dramatic reduction in activity due to chemical reactions involved in enzyme immobilization, multiple-point binding, etc., and the extent of activity reduction is a function of membrane hydrophilicity (e.g. activity in cellulosic membrane >> polysulfone membrane). We have used molecular biology to effect site-specific immobilization of enzymes in a manner that orients the active site away from the polymeric membrane surface, thus resulting in higher enzyme activity that approaches that in solution and in increased stability of the enzyme relative to the enzyme in solution. A prediction of this site-specific method of enzyme immobilization, which in this study with subtilisin and organophosphorus hydrolase consists of a fusion tag genetically added to these enzymes and subsequent immobilization via the anti-tag antibody and membrane-bound protein A, is that the active site conformation will more closely resemble that of the enzyme in solution than is the case for random immobilization. This hypothesis was confirmed using a new electron paramagnetic resonance (EPR) spin label active site titration method that determines the amount of spin label bound to the active site of the immobilized enzyme. This value nearly perfectly matched the enzyme activity, and the results suggested: (a) a spectroscopic method for measuring activity and thus the extent of active enzyme immobilization in membrane, which may have advantages in cases where optical methods can not be used due to light scattering interference; (b) higher spin label incorporation (and hence activity) in enzymes that had been site-specifically immobilized versus random immobilization; (c) higher spin label incorporation in enzymes immobilized onto hydrophilic

  3. Immobilization of inorganic pyrophosphatase on nanodiamond particles retaining its high enzymatic activity.

    Science.gov (United States)

    Rodina, Elena V; Valueva, Anastasiya V; Yakovlev, Ruslan Yu; Vorobyeva, Nataliya N; Kulakova, Inna I; Lisichkin, Georgy V; Leonidov, Nikolay B

    2015-12-21

    Nanodiamond (ND) particles are popular platforms for the immobilization of molecular species. In the present research, enzyme Escherichia coli inorganic pyrophosphatase (PPase) was immobilized on detonation ND through covalent or noncovalent bonding and its enzymatic activity was characterized. Factors affecting adsorption of PPase such as ND size and surface chemistry were studied. The obtained material is a submicron size association of ND particles and protein molecules in approximately equal amounts. Both covalently and noncovalently immobilized PPase retains a significant enzymatic activity (up to 95% of its soluble form) as well as thermostability. The obtained hybrid material has a very high enzyme loading capacity (∼1 mg mg(-1)) and may be considered as a promising delivery system of biologically active proteinaceous substances, particularly in the treatment of diseases such as calcium pyrophosphate crystal deposition disease and related pathologies. They can also be used as recoverable heterogeneous catalysts in the traditional uses of PPase.

  4. Characteristics of Immobilized Urease on Grafted Alginate Bead Systems

    Directory of Open Access Journals (Sweden)

    Enas N. Danial

    2015-04-01

    Full Text Available This study evaluated the biological importance of immobilized urease enzyme over the free urease. The support material used for urease immobilization was alginate. Generally, the immobilization of urease in alginate gel showed a marked increase in Km and Vmax. However, the immobilized urease showed higher thermal stability than that of free enzyme. The rate of thermal inactivation of the immobilized enzyme decreased due to entrapment in gel matrix. Also, the activity of the immobilized urease was more stable in retention than that of the free enzyme during the storage in solution, although the activity of the immobilized enzyme was lower in comparison with the free enzyme. A stable immobilized system and long storage life are convenient for applications that would not be feasible with a soluble enzyme system. These results highlighted the technical and biochemical benefits of immobilized urease over the free enzyme.

  5. Improvement of the stability and activity of immobilized glucose oxidase on modified iron oxide magnetic nanoparticles

    Science.gov (United States)

    Abbasi, Mahboube; Amiri, Razieh; Bordbar, Abdol-Kalegh; Ranjbakhsh, Elnaz; Khosropour, Ahmad-Reza

    2016-02-01

    Immobilized proteins and enzymes are widely investigated in the medical field as well as the food and environmental fields. In this study, glucose oxidase (GOX) was covalently immobilized on the surface of modified iron oxide magnetic nanoparticles (MIMNs) to produce a bioconjugate complex. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to the size, shape and structure characterization of the MIMNs. Binding of GOX to these MIMNs was confirmed by using FT-IR spectroscopy. The stability of the immobilized and free enzyme at different temperature and pH values was investigated by measuring the enzymatic activity. These studies reveal that the enzyme's stability is enhanced by immobilization. Further experiments showed that the storage stability of the enzyme is improved upon binding to the MIMNs. The results of kinetic measurements suggest that the effect of the immobilization process on substrate and product diffusion is small. Such bioconjugates can be considered as a catalytic nanodevice for accelerating the glucose oxidation reaction for biotechnological purposes.

  6. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    International Nuclear Information System (INIS)

    Jin Xin; Li Jufang; Huang Pingying; Dong Xuyan; Guo Lulu; Yang Liang; Cao Yuancheng; Wei Fang; Zhao Yuandi

    2010-01-01

    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63±2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  7. Immobilization of Murine Anti-BMP-2 Monoclonal Antibody on Various Biomaterials for Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Sahar Ansari

    2014-01-01

    Full Text Available Biomaterials are widely used as scaffolds for tissue engineering. We have developed a strategy for bone tissue engineering that entails application of immobilized anti-BMP-2 monoclonal antibodies (mAbs to capture endogenous BMPs in vivo and promote antibody-mediated osseous regeneration (AMOR. The purpose of the current study was to compare the efficacy of immobilization of a specific murine anti-BMP-2 mAb on three different types of biomaterials and to evaluate their suitability as scaffolds for AMOR. Anti-BMP-2 mAb or isotype control mAb was immobilized on titanium (Ti microbeads, alginate hydrogel, and ACS. The treated biomaterials were surgically implanted in rat critical-sized calvarial defects. After 8 weeks, de novo bone formation was assessed using micro-CT and histomorphometric analyses. Results showed de novo bone regeneration with all three scaffolds with immobilized anti-BMP-2 mAb, but not isotype control mAb. Ti microbeads showed the highest volume of bone regeneration, followed by ACS. Alginate showed the lowest volume of bone. Localization of BMP-2, -4, and -7 antigens was detected on all 3 scaffolds with immobilized anti-BMP-2 mAb implanted in calvarial defects. Altogether, these data suggested a potential mechanism for bone regeneration through entrapment of endogenous BMP-2, -4, and -7 proteins leading to bone formation using different types of scaffolds via AMOR.

  8. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    Energy Technology Data Exchange (ETDEWEB)

    Jin Xin [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Li Jufang [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Huang Pingying [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Dong Xuyan [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Guo Lulu [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Yang Liang; Cao Yuancheng [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Wei Fang [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Zhao Yuandi, E-mail: zydi@mail.hust.edu.c [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China)

    2010-07-15

    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63+-2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  9. Immobilization of biomolecules on cysteamine-modified polyaniline film for highly sensitive biosensing.

    Science.gov (United States)

    Cai, Qi; Xu, Baojian; Ye, Lin; Di, Zengfeng; Zhang, Jishen; Jin, Qinghui; Zhao, Jianlong; Xue, Jian; Chen, Xianfeng

    2014-03-01

    We present a new cysteamine (CS)-modified polyaniline (PANI) film for highly efficient immobilization of biomolecules in biosensing technology. This electrochemical deposited PANI film treated with CS and glutaraldehyde could be employed as an excellent substrate for biomolecules immobilization. The parameters of PANI growth were optimized to obtain suitable surface morphology of films for biomolecules combination with the help of electron and atomic force microscopy. Cyclic voltammetry (CV) was utilized to illustrate the different electrochemical activities of each modified electrode. Due to the existence of sulfydryl group and amino group in CS, surface modification with CS was proven to reduce oxidized units on PANI film remarkably, as evidenced by both ATR-FTIR and Raman spectroscopy characterizations. Furthermore, bovine serum albumin (BSA) was used as the model protein to investigate the immobilization efficiency of biomolecules on the PANI film, comparative study using quartz crystal microbalance (QCM) showed that BSA immobilized on CS-modified PANI could be increased by at least 20% than that without CS-modified PANI in BSA solution with the concentration of 0.1-1mg/mL. The CS-modified PANI film would be significant for the immobilization and detection of biomolecules and especially promising in the application of immunosensor for ultrasensitive detection. © 2013 Published by Elsevier B.V.

  10. [Comparison of fibroblastic cell compatibility of type I collagen-immobilized titanium between electrodeposition and immersion].

    Science.gov (United States)

    Kyuragi, Takeru

    2014-03-01

    Titanium is widely used for medical implants. While many techniques for surface modification have been studied for optimizing its biocompatibility with hard tissues, little work has been undertaken to explore ways of maximizing its biocompatibility with soft tissues. We investigated cell attachment to titanium surfaces modified with bovine Type I collagen immobilized by either electrodeposition or a conventional immersion technique. The apparent thickness and durability of the immobilized collagen layer were evaluated prior to incubation of the collagen-immobilized titanium surfaces with NIH/3T3 mouse embryonic fibroblasts. The initial cell attachment and expression of actin and vinculin were evaluated. We determined that the immobilized collagen layer was much thicker and more durable when placed using the electrodeposition technique than the immersion technique. Both protocols produced materials that promoted better cell attachment, growth and structural protein expression than titanium alone. However, electrodeposition was ultimately superior to immersion because it is quicker to perform and produces a more durable collagen coating. We conclude that electrodeposition is an effective technique for immobilizing type I collagen on titanium surfaces, thus improving their cytocompatibility with fibroblasts.

  11. Inorganic Materials as Supports for Covalent Enzyme Immobilization: Methods and Mechanisms

    Directory of Open Access Journals (Sweden)

    Paolo Zucca

    2014-09-01

    Full Text Available Several inorganic materials are potentially suitable for enzymatic covalent immobilization, by means of several different techniques. Such materials must meet stringent criteria to be suitable as solid matrices: complete insolubility in water, reasonable mechanical strength and chemical resistance under the operational conditions, the capability to form manageable particles with high surface area, reactivity towards derivatizing/functionalizing agents. Non-specific protein adsorption should be always considered when planning covalent immobilization on inorganic solids. A huge mass of experimental work has shown that silica, silicates, borosilicates and aluminosilicates, alumina, titania, and other oxides, are the materials of choice when attempting enzyme immobilizations on inorganic supports. More recently, some forms of elemental carbon, silicon, and certain metals have been also proposed for certain applications. With regard to the derivatization/functionalization techniques, the use of organosilanes through silanization is undoubtedly the most studied and the most applied, although inorganic bridge formation and acylation with selected acyl halides have been deeply studied. In the present article, the most common inorganic supports for covalent immobilization of the enzymes are reviewed, with particular focus on their advantages and disadvantages in terms of enzyme loadings, operational stability, undesired adsorption, and costs. Mechanisms and methods for covalent immobilization are also discussed, focusing on the most widespread activating approaches (such as glutaraldehyde, cyanogen bromide, divinylsulfone, carbodiimides, carbonyldiimidazole, sulfonyl chlorides, chlorocarbonates, N-hydroxysuccinimides.

  12. Immobilization of Lipase from Geobacillus sp. and Its Application in Synthesis of Methyl Salicylate.

    Science.gov (United States)

    Bhardwaj, Kamal Kumar; Saun, Nitin Kumar; Gupta, Reena

    2017-04-03

    The present study showed unique properties of an alkaline, thermophilic lipase of Geobacillus sp. which was isolated from soil of hot spring. The study was aimed to investigate the optimum immobilization conditions of lipase onto silica gel matrix (100-200 mesh) by surface adsorption method and its application in the synthesis of methyl salicylate. Lipase immobilized by surface adsorption onto silica pretreated with 4% glutaraldehyde showed 74.67% binding of protein and the optimum binding time for glutaraldehyde was found to be 2 h. The enzyme showed maximum activity at temperature 55°C, incubation time of 10 min at pH 9.5 of Tris buffer (0.1 M). Free as well as immobilized lipase was more specific to p-NPP (20 mM). All the metal ions and detergents used had inhibitory effect on free as well as immobilized enzyme. The silica immobilized enzyme was reused for hydrolysis and it retained almost 40.78% of its original activity up to 4 th cycle. On optimizing different parameters such as molar ratio, incubation time, temperature, amount of enzyme, amount of molecular sieve, the % yield of methyl salicylate was found to be 82.94.

  13. Lipase immobilized on the hydrophobic polytetrafluoroethene membrane with nonwoven fabric and its application in intensifying synthesis of butyl oleate.

    Science.gov (United States)

    Wang, Shu-Guang; Zhang, Wei-Dong; Li, Zheng; Ren, Zhong-Qi; Liu, Hong-Xia

    2010-11-01

    The synthesis of butyl oleate was studied in this paper with immobilized lipase. Five types of membrane were used as support to immobilize Rhizopus arrhizus lipase by following a procedure combining filtration and protein cross-linking. Results showed that hydrophobic polytetrafluoroethene membrane with nonwoven fabric (HO-PTFE-NF) was the favorite choice in terms of higher protein loading, activity, and specific activity of immobilized lipase. The factors including solvent polarity, lipase dosage, concentration, and molar ratio of substrate and temperature were found to have significant influence on conversion. Results showed that hexane (logP = 3.53) was a favorable solvent for the biosynthesis of butyl oleate in our studies. The optimal conditions were experimentally determined of 50 U immobilized lipase, molar ratio of oleic acid to butanol of 1.0, substrate concentration of 0.12 mol/L, temperature of 37 °C, and reaction time of 2 h. The conversion was beyond 91% and decreased slightly after 18 cycles. Lipase immobilization can improve the conversion and the repeated use of immobilized lipase relative to free lipase.

  14. Plasma membrane isolation using immobilized concanavalin A magnetic beads.

    Science.gov (United States)

    Lee, Yu-Chen; Srajer Gajdosik, Martina; Josic, Djuro; Lin, Sue-Hwa

    2012-01-01

    Isolation of highly purified plasma membranes is the key step in constructing the plasma membrane proteome. Traditional plasma membrane isolation method takes advantage of the differential density of organelles. While differential centrifugation methods are sufficient to enrich for plasma membranes, the procedure is lengthy and results in low recovery of the membrane fraction. Importantly, there is significant contamination of the plasma membranes with other organelles. The traditional agarose affinity matrix is suitable for isolating proteins but has limitation in separating organelles due to the density of agarose. Immobilization of affinity ligands to magnetic beads allows separation of affinity matrix from organelles through magnets and could be developed for the isolation of organelles. We have developed a simple method for isolating plasma membranes using lectin concanavalin A (ConA) magnetic beads. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. The ConA magnetic beads are used to bind glycosylated proteins present in the membranes. The bound membranes are solubilized from the magnetic beads with a detergent containing the competing sugar alpha methyl mannoside. In this study, we describe the procedure of isolating rat liver plasma membranes using sucrose density gradient centrifugation as described by Neville. We then further purify the membrane fraction by using ConA magnetic beads. After this purification step, main liver plasma membrane proteins, especially the highly glycosylated ones and proteins containing transmembrane domains could be identified by LC-ESI-MS/MS. While not described here, the magnetic bead method can also be used to isolate plasma membranes from cell lysates. This membrane purification method should expedite the cataloging of plasma membrane proteome.

  15. Tentacle carrier for immobilization of potato phenoloxidase and its application for halogenophenols removal from aqueous solutions

    International Nuclear Information System (INIS)

    Lončar, Nikola; Vujčić, Zoran

    2011-01-01

    Highlights: ► We synthesized novel immobilization carrier from reused DEAE-cellulose. ► We used it for immobilization of PPO through coordinative bonding with copper ions. ► Immobilized PPO showed better characteristics than soluble PPO. ► TC-PPO removed over 90% of halogenophenols at concentration of 100 mg/L. - Abstract: Halogenated compounds represent one of the most dangerous environmental pollutants, due to their widespread usage as biocides, fungicides, disinfectants, solvent and other industrial chemicals. Immobilization of a protein through coordinate bonds formed with divalent metal ions is becoming an attractive method due to its reversible nature, since the protein may be easily removed from the support matrix through interruption of the protein–metal bond hence giving inherently cleaner and cheaper technology for wastewater treatment. We have synthesized novel ‘tentacle’ carrier (TC) and used it for immobilization of partially purified potato polyphenol oxidase (PPO). The obtained biocatalyst TC-PPO showed pH optimum at 7.0–8.0 and temperature optimum at 25 °C. Immobilized PPO shows almost 100% of activity at 0 °C. TC-PPO was more resistant to the denaturation induced by sodium dodecyl sulphate (SDS) detergent as compared to its soluble counterpart and was even slightly activated at SDS concentration of 1%. TC-PPO was tested in the batch reactor for 4-chlorophenol and 4-bromophenol removal. More than 90% removal was achieved for both halogenophenols at concentration of 100 mg/L from aqueous solution. For both halogenophenols TC-PPO works with over 90% removal during first three cycles which decrease to 60% removal efficiency after six cycles each of 8 h duration.

  16. Excess Weapons Plutonium Immobilization in Russia

    International Nuclear Information System (INIS)

    Jardine, L.; Borisov, G.B.

    2000-01-01

    The joint goal of the Russian work is to establish a full-scale plutonium immobilization facility at a Russian industrial site by 2005. To achieve this requires that the necessary engineering and technical basis be developed in these Russian projects and the needed Russian approvals be obtained to conduct industrial-scale immobilization of plutonium-containing materials at a Russian industrial site by the 2005 date. This meeting and future work will provide the basis for joint decisions. Supporting R and D projects are being carried out at Russian Institutes that directly support the technical needs of Russian industrial sites to immobilize plutonium-containing materials. Special R and D on plutonium materials is also being carried out to support excess weapons disposition in Russia and the US, including nonproliferation studies of plutonium recovery from immobilization forms and accelerated radiation damage studies of the US-specified plutonium ceramic for immobilizing plutonium. This intriguing and extraordinary cooperation on certain aspects of the weapons plutonium problem is now progressing well and much work with plutonium has been completed in the past two years. Because much excellent and unique scientific and engineering technical work has now been completed in Russia in many aspects of plutonium immobilization, this meeting in St. Petersburg was both timely and necessary to summarize, review, and discuss these efforts among those who performed the actual work. The results of this meeting will help the US and Russia jointly define the future direction of the Russian plutonium immobilization program, and make it an even stronger and more integrated Russian program. The two objectives for the meeting were to: (1) Bring together the Russian organizations, experts, and managers performing the work into one place for four days to review and discuss their work with each other; and (2) Publish a meeting summary and a proceedings to compile reports of all the

  17. Disposition of surplus fissile materials via immobilization

    International Nuclear Information System (INIS)

    Gray, L.W.; Kan, T.; Sutcliffe, W.G.; McKibben, J.M.; Danker, W.

    1995-01-01

    In the Cold War aftermath, the US and Russia have agreed to large reductions in nuclear weapons. To aid in the selection of long-term management options, the USDOE has undertaken a multifaceted study to select options for storage and disposition of surplus plutonium (Pu). One disposition alternative being considered is immobilization. Immobilization is a process in which surplus Pu would be embedded in a suitable material to produce an appropriate form for ultimate disposal. To arrive at an appropriate form, we first reviewed published information on HLW immobilization technologies to identify forms to be prescreened. Surviving forms were screened using multi-attribute utility analysis to determine promising technologies for Pu immobilization. We further evaluated the most promising immobilization families to identify and seek solutions for chemical, chemical engineering, environmental, safety, and health problems; these problems remain to be solved before we can make technical decisions about the viability of using the forms for long-term disposition of Pu. All data, analyses, and reports are being provided to the DOE Office of Fissile Materials Disposition to support the Record of Decision that is anticipated in Summer of 1996

  18. Production of cellulase from immobilized Trichoderma reesei

    International Nuclear Information System (INIS)

    Kasai, Noboru; Tamada, Masao; Kumakura, Minoru

    1989-05-01

    This report completed the results that obtained on the study of the enzyme activity in the culture of immobilized Trichoderma reesei cells in flask scale (100ml) and bench scale (30l). In the flask scale culture, the batch and repeated batch culture were carried out, and in the bench scale culture, the batch, repeated batch and continuous culture were done by using a culture equipment that is an unit process of the bench scale test plant for saccharification of cellulosic wastes. The enzyme activity of the immobilized cells was higher than that of the intact cells in the flask scale culture and it was confirmed that the enzyme activity was not decreased on the repeated batch culture of six times even. In the bench scale culture, it was found that a optimum culture condition of the immobilized cells was not different from that of the free cells and the immobilized cells gave the enzyme solution with a high enzyme activity in the culture condition of 450rpm stirring speed and air supply of 0.1v/v/m above. The technique of the repeated batch and continuous culture for long times in bench scale without contamination was established. The enzyme activity of the immobilized cells in continuous culture became to be 85 % to that in batch culture and it was found that the enzyme solution with high enzyme activity was continuously obtained in the continuous culture for long times. (author)

  19. Delayed recovery of skeletal muscle mass following hindlimb immobilization in mTOR heterozygous mice.

    Directory of Open Access Journals (Sweden)

    Susan M Lang

    Full Text Available The present study addressed the hypothesis that reducing mTOR, as seen in mTOR heterozygous (+/- mice, would exaggerate the changes in protein synthesis and degradation observed during hindlimb immobilization as well as impair normal muscle regrowth during the recovery period. Atrophy was produced by unilateral hindlimb immobilization and data compared to the contralateral gastrocnemius. In wild-type (WT mice, the gradual loss of muscle mass plateaued by day 7. This response was associated with a reduction in basal protein synthesis and development of leucine resistance. Proteasome activity was consistently elevated, but atrogin-1 and MuRF1 mRNAs were only transiently increased returning to basal values by day 7. When assessed 7 days after immobilization, the decreased muscle mass and protein synthesis and increased proteasome activity did not differ between WT and mTOR(+/- mice. Moreover, the muscle inflammatory cytokine response did not differ between groups. After 10 days of recovery, WT mice showed no decrement in muscle mass, and this accretion resulted from a sustained increase in protein synthesis and a normalization of proteasome activity. In contrast, mTOR(+/- mice failed to fully replete muscle mass at this time, a defect caused by the lack of a compensatory increase in protein synthesis. The delayed muscle regrowth of the previously immobilized muscle in the mTOR(+/- mice was associated with a decreased raptor•4EBP1 and increased raptor•Deptor binding. Slowed regrowth was also associated with a sustained inflammatory response (e.g., increased TNFα and CD45 mRNA during the recovery period and a failure of IGF-I to increase as in WT mice. These data suggest mTOR is relatively more important in regulating the accretion of muscle mass during recovery than the loss of muscle during the atrophy phase, and that protein synthesis is more sensitive than degradation to the reduction in mTOR during muscle regrowth.

  20. Delayed recovery of skeletal muscle mass following hindlimb immobilization in mTOR heterozygous mice.

    Science.gov (United States)

    Lang, Susan M; Kazi, Abid A; Hong-Brown, Ly; Lang, Charles H

    2012-01-01

    The present study addressed the hypothesis that reducing mTOR, as seen in mTOR heterozygous (+/-) mice, would exaggerate the changes in protein synthesis and degradation observed during hindlimb immobilization as well as impair normal muscle regrowth during the recovery period. Atrophy was produced by unilateral hindlimb immobilization and data compared to the contralateral gastrocnemius. In wild-type (WT) mice, the gradual loss of muscle mass plateaued by day 7. This response was associated with a reduction in basal protein synthesis and development of leucine resistance. Proteasome activity was consistently elevated, but atrogin-1 and MuRF1 mRNAs were only transiently increased returning to basal values by day 7. When assessed 7 days after immobilization, the decreased muscle mass and protein synthesis and increased proteasome activity did not differ between WT and mTOR(+/-) mice. Moreover, the muscle inflammatory cytokine response did not differ between groups. After 10 days of recovery, WT mice showed no decrement in muscle mass, and this accretion resulted from a sustained increase in protein synthesis and a normalization of proteasome activity. In contrast, mTOR(+/-) mice failed to fully replete muscle mass at this time, a defect caused by the lack of a compensatory increase in protein synthesis. The delayed muscle regrowth of the previously immobilized muscle in the mTOR(+/-) mice was associated with a decreased raptor•4EBP1 and increased raptor•Deptor binding. Slowed regrowth was also associated with a sustained inflammatory response (e.g., increased TNFα and CD45 mRNA) during the recovery period and a failure of IGF-I to increase as in WT mice. These data suggest mTOR is relatively more important in regulating the accretion of muscle mass during recovery than the loss of muscle during the atrophy phase, and that protein synthesis is more sensitive than degradation to the reduction in mTOR during muscle regrowth.

  1. Immobilization of oxidases and their analytical applications

    International Nuclear Information System (INIS)

    Yasinzai, M.

    2007-01-01

    Immobilized enzymes are replacing their soluble counter-parts in nearly every field of application. These enzyme modifications have evolved from a research curiosity into an entire branch of Biotechnology. An immobilization method for flavin containing oxidases and their use in flow injection system is described. An electrochemical detector for H/sub 2/O/sub 2/ is assembled which is used effectively for the determination of glucose using more common glucose oxidase and the simultaneous determination of sugars. The combination of oxidases with hydrolases have been used for the determination of maltose and starch. (author)

  2. A disposal centre for immobilized nuclear waste

    International Nuclear Information System (INIS)

    1980-02-01

    This report describes a conceptual design of a disposal centre for immobilized nuclear waste. The surface facilities consist of plants for the preparation of steel cylinders containing nuclear waste immobilized in glass, shaft headframe buildings and all necessary support facilities. The underground disposal vault is located on one level at a depth of 1000 m. The waste cylinders are emplaced into boreholes in the tunnel floors. All surface and subsurface facilities are described, operations and schedules are summarized, and cost estimates and manpower requirements are given. (auth)

  3. Radiation Synthesis of Nanogel for Bioactives Immobilization

    Energy Technology Data Exchange (ETDEWEB)

    Hamzah, M. Y. [Polymer Modification Group, Malaysian Nuclear Agency, Bangi (Malaysia)

    2009-07-01

    Both hydrophilic and hydrophobic core nanogel are currently being developed for immobilization and delivery purposes in Malaysian Nuclear Agency. Hydrophilic nanogel is produced by using inverse micelles irradiation of polyethelyne glycol diacrylate (PEGDA). The hydrophobic nanogel is produced via irradiation of acrylated form of palm oil. These nanogels will be used to immobilize bio actives such as curcumin, tyhmoquinone, oryzanol and chitosan. Preliminary investigation of the nanogel size using dynamic light scattering (DLS) shows that nanogel with sizes below 100nm can be obtained. (author)

  4. Plutonium Immobilization Can Loading Conceptual Design

    International Nuclear Information System (INIS)

    Kriikku, E.

    1999-01-01

    'The Plutonium Immobilization Facility will encapsulate plutonium in ceramic pucks and seal the pucks inside welded cans. Remote equipment will place these cans in magazines and the magazines in a Defense Waste Processing Facility (DWPF) canister. The DWPF will fill the canister with glass for permanent storage. This report discusses the Plutonium Immobilization can loading conceptual design and includes a process block diagram, process description, preliminary equipment specifications, and several can loading issues. This report identifies loading pucks into cans and backfilling cans with helium as the top priority can loading development areas.'

  5. Radiation Synthesis of Nanogel for Bioactives Immobilization

    International Nuclear Information System (INIS)

    Hamzah, M.Y.

    2009-01-01

    Both hydrophilic and hydrophobic core nanogel are currently being developed for immobilization and delivery purposes in Malaysian Nuclear Agency. Hydrophilic nanogel is produced by using inverse micelles irradiation of polyethelyne glycol diacrylate (PEGDA). The hydrophobic nanogel is produced via irradiation of acrylated form of palm oil. These nanogels will be used to immobilize bio actives such as curcumin, tyhmoquinone, oryzanol and chitosan. Preliminary investigation of the nanogel size using dynamic light scattering (DLS) shows that nanogel with sizes below 100nm can be obtained. (author)

  6. Plutonium Immobilization Can Loading Conceptual Design

    Energy Technology Data Exchange (ETDEWEB)

    Kriikku, E.

    1999-05-13

    'The Plutonium Immobilization Facility will encapsulate plutonium in ceramic pucks and seal the pucks inside welded cans. Remote equipment will place these cans in magazines and the magazines in a Defense Waste Processing Facility (DWPF) canister. The DWPF will fill the canister with glass for permanent storage. This report discusses the Plutonium Immobilization can loading conceptual design and includes a process block diagram, process description, preliminary equipment specifications, and several can loading issues. This report identifies loading pucks into cans and backfilling cans with helium as the top priority can loading development areas.'

  7. Immobilization of spent resin with epoxy resin

    International Nuclear Information System (INIS)

    Gultom, O.; Suryanto; Sayogo; Ramdan

    1997-01-01

    immobilization of spent resin using epoxy resin has been conducted. The spent resin was mixtured with epoxy resin in variation of concentration, i.e., 30, 40, 50, 60, 70 weight percent of spent resin. The mixture were pour into the plastic tube, with a diameter of 40 mm and height of 40 mm. The density, compressive strength and leaching rate were respectively measured by quanta chrome, paul weber apparatus and gamma spectrometer. The results showed that the increasing of waste concentration would be decreased the compressive strength, and increased density by immobilized waste. The leaching rate of 137 Cs from waste product was not detected in experiment (author)

  8. Evaluation of conformity of a first prescription of lipid-based formulation of amphotericin B in a university-teaching pediatric hospital.

    Science.gov (United States)

    Agogué, Claire; Bussières, Jean-François; Dehaut, Catherine; Lebel, Denis; Brochet, Marie-Sophie

    2006-04-01

    Invasive fungal infections are an important cause of morbidity and mortality in immunodeficient children. Amphotericin B is an important therapeutic agent for the treatment of invasive fungal infections but is associated with significant toxicities and high acquisition costs. The purpose of this study was to evaluate physician adherence to a local guideline for the use of lipid-based amphotericin B. The study was approved through Pharmacology & Therapeutics (P&T) committee activities. A retrospective drug utilization review (DUR) was conducted. All orders written between January 1, 2003, and December 31, 2004, were reviewed. Demographic and descriptive clinical data were collected as well as variables related to the drug order process. Conformity rates were calculated for the primary objective criteria (authorized prescribers - infectious disease members; recommended drug of choice-Abelcet; accepted indications; and presence of underlying conditions). A total of 109 orders for 70 patients were reviewed by a single research assistant for a 2-year period. Global conformity rate for all four criteria was calculated at 7.3%. Non-conformity was mostly associated with the absence of underlying conditions (e.g., prerenal insufficiency or presence of nephrotoxicity due to amphotericin B desoxycholate) in 84.5% of the cases. Infusionrelated adverse drug reactions partly explained a switch to a non-formulary lipid-based amphotericin B product. External factors (newly published results since the adoption of the guideline and continuous marketing practices) and internal factors (availability of non-formulary process, inefficient DUR process) could have contributed to non-adherence to a local guideline. This study shows low adherence to P&T committee drug guidelines on lipid-based amphotericin B. Continuous and efficient DUR processes should be in place to monitor drug guideline adherence.

  9. Direct electrochemistry and electrocatalysis of myoglobin immobilized on zirconia/multi-walled carbon nanotube nanocomposite

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Ruping; Deng, Minqiang; Cui, Sanguan; Chen, Hong [Department of Chemistry and Institute for Advanced Study, Nanchang University, Nanchang 330031 (China); Qiu, Jianding, E-mail: jdqiu@ncu.edu.cn [Department of Chemistry and Institute for Advanced Study, Nanchang University, Nanchang 330031 (China)

    2010-12-15

    Zirconia/multi-walled carbon nanotube (ZrO{sub 2}/MWCNT) nanocomposite was prepared by hydrothermal treatment of MWCNTs in ZrOCl{sub 2}.8H{sub 2}O aqueous solution. The morphology and structure of the synthesized ZrO{sub 2}/MWCNT nanocomposite were characterized by transmission electron microscopy and X-ray diffraction analysis. It was found that ZrO{sub 2} nanoparticles homogeneously distributed on the sidewall of MWCNTs. Myoglobin (Mb), as a model protein to investigate the nanocomposite, was immobilized on ZrO{sub 2}/MWCNT nanocomposite. Ultraviolet-visible spectroscopy and electrochemical measurements showed that the nanocomposite could retain the bioactivity of the immobilized Mb to a large extent. The Mb immobilized in the composite showed excellent direct electrochemistry and electrocatalytic activity to the reduction of hydrogen peroxide (H{sub 2}O{sub 2}). The linear response range of the biosensor to H{sub 2}O{sub 2} concentration was from 1.0 to 116.0 {mu}M with the limit of detection of 0.53 {mu}M (S/N = 3). The ZrO{sub 2}/MWCNT nanocomposite provided a good biocompatible matrix for protein immobilization and biosensors preparation.

  10. Direct electrochemistry of horseradish peroxidase immobilized on electrografted 4-ethynylphenyl film via click chemistry

    International Nuclear Information System (INIS)

    Ran Qin; Peng Ru; Liang Cong; Ye Siqiu; Xian Yuezhong; Zhang Wenjing; Jin Litong

    2011-01-01

    Graphical abstract: Hydrogen peroxide biosensor was developed based on electrochemically assisted aryldiazonium salt chemistry and click chemistry. Highlights: → A simple, versatile two-step approach, which is based on electrochemically assisted aryldiazonium salt chemistry and Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction has been developed for covalent redox proteins immobilization and biosensing for the first time. In this work, azido group modified HRP was covalently grafted on 4-ethylnylphenyl diazonium compound via CuAAC reaction and a novel electrochemical hydrogen peroxide biosensor was successfully fabricated. - Abstract: In this paper, a simple two-step approach for redox protein immobilization was introduced. Firstly, alkynyl-terminated film was formed on electrode surface by electrochemical reduction of 4-ethylnylphenyl (4-EP) diazonium compound. Then, horseradish peroxidase (HRP) modified with azido group was covalently immobilized onto the electrografted film via click reaction. Reflection absorption infrared (RAIR) spectroscopy and electrochemical methods were used to characterize the modification process. The results indicate that HRP retains its native structure and shows fast direct electron transfer. Moreover, the immobilized HRP shows excellent electrocatalytic reduction activity toward H 2 O 2 with a linear range of 5.0 x 10 -6 to 9.3 x 10 -4 mol L -1 .

  11. Direct electrochemistry of horseradish peroxidase immobilized on electrografted 4-ethynylphenyl film via click chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Ran Qin; Peng Ru; Liang Cong; Ye Siqiu [Department of Chemistry, East China Normal University, Shanghai 200062 (China); Xian Yuezhong, E-mail: yzxian@chem.ecnu.edu.cn [Department of Chemistry, East China Normal University, Shanghai 200062 (China); Zhang Wenjing; Jin Litong [Department of Chemistry, East China Normal University, Shanghai 200062 (China)

    2011-07-04

    Graphical abstract: Hydrogen peroxide biosensor was developed based on electrochemically assisted aryldiazonium salt chemistry and click chemistry. Highlights: > A simple, versatile two-step approach, which is based on electrochemically assisted aryldiazonium salt chemistry and Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction has been developed for covalent redox proteins immobilization and biosensing for the first time. In this work, azido group modified HRP was covalently grafted on 4-ethylnylphenyl diazonium compound via CuAAC reaction and a novel electrochemical hydrogen peroxide biosensor was successfully fabricated. - Abstract: In this paper, a simple two-step approach for redox protein immobilization was introduced. Firstly, alkynyl-terminated film was formed on electrode surface by electrochemical reduction of 4-ethylnylphenyl (4-EP) diazonium compound. Then, horseradish peroxidase (HRP) modified with azido group was covalently immobilized onto the electrografted film via click reaction. Reflection absorption infrared (RAIR) spectroscopy and electrochemical methods were used to characterize the modification process. The results indicate that HRP retains its native structure and shows fast direct electron transfer. Moreover, the immobilized HRP shows excellent electrocatalytic reduction activity toward H{sub 2}O{sub 2} with a linear range of 5.0 x 10{sup -6} to 9.3 x 10{sup -4} mol L{sup -1}.

  12. Potential antioxidant peptides produced from whey hydrolysis with an immobilized aspartic protease from Salpichroa origanifolia fruits.

    Science.gov (United States)

    Rocha, Gabriela Fernanda; Kise, Francisco; Rosso, Adriana Mabel; Parisi, Mónica Graciela

    2017-12-15

    An aspartic protease from Salpichroa origanifolia fruits was successfully immobilized onto an activated support of glutaraldehyde agarose. The immobilized enzyme presented higher thermal stability than the free enzyme from 40°C to 50°C and high reusability, retaining 54% of the initial activity after ten cycles of the process. Whey protein concentrates (WPC) were hydrolyzed with both free and immobilized enzyme, reaching a similar degree of hydrolysis of approximately 6-8% after 20h. In addition, the immobilized derivate hydrolyzed α-lactalbumin protein with a higher affinity than β-lactoglobulin. The hydrolysate was ultra-filtrated, and the fractions were evaluated for antioxidant activities with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity method. The fraction containing peptides with a molecular mass below 3kDa demonstrated a strong radical quenching effect (IC 50: 0.48mg/ml). These results suggest that hydrolyzed WPC could be considered as a promising source of natural food antioxidants for the development of functional food. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. The immobilization of filamentous fungi by cellular adherence and surface film growth on radiopolymerized hydrogels

    International Nuclear Information System (INIS)

    Petre, M.; Zarnea, G.; Adrian, P.; Gheorghiu, E.; Gheordunescu, V.

    1998-01-01

    The continuous biodegradation processes of cellulose wastes, from wine-producing industry and canned food processing were performed using immobilized fungal cells on complex polyhydrogels such as: collagen-polyacrylamide (CPAA), elastin-polyacrylamide (EPAA), gelatin-polyacrylamide (GPAA) and poly-hydroxy-ethyl-methyl-methacrylate (PHEMA) which were prepared by experimental radiopolymerization methods using a 60 Co radioactive source. The experiments were performed using cellulolytic micro- and macrofungal species and the immobilization methods were applied in connection with morphogenetic characteristics of each one of these species that were used as inoculum. A flow-cascade bioreactor was achieved as a research laboratory tool in order to control the cellulose waste biodegradation in continuous processes using immobilized fungal cells on complex polyhydrogels. In this way the continuous control was possible for fungal metabolic activity during the cellulose biodegradation processes by microscopical hyphal growth measurements inside the polyhydrogels and, also, by mycelial protein content analysis. The tested fungal species were Pleurotus ostreatus and Pleurotus florida from macromycetes and Trichoderma viride from micromycetes. There were noticed significant differences in biodegradation rate of cellulose wastes between 'in vitro' immobilized fungal cultures and free fungal cells cultures used as control samples to be compared with cellulose biodegradation processes in natural conditions of polluted ecosystems. The fungal growth displayed by soluble mycelial protein were compared with cellulose biodegradation rates which were expressed by cellulose weight loss in the culture medium. (authors)

  14. Toward the Establishment of Standardized In Vitro Tests for Lipid-Based Formulations. 5. Lipolysis of Representative Formulations by Gastric Lipase

    DEFF Research Database (Denmark)

    Bakala-N'Goma, Jean-Claude; Williams, Hywel D.; Sassene, Philip J.

    2015-01-01

    Purpose Lipid-based formulations (LBF) are substrates for digestive lipases and digestion can significantly alter their properties and potential to support drug absorption. LBFs have been widely examined for their behaviour in the presence of pancreatic enzymes. Here, the impact of gastric lipase...... on the digestion of representative formulations from the Lipid Formulation Classification System has been investigated. Methods The pHstat technique was used to measure the lipolysis by recombinant dog gastric lipase (rDGL) of eight LBFs containing either medium (MC) or long (LC) chain triglycerides and a range...

  15. Preparation of Laccase Immobilized Cryogels and Usage for Decolorization

    Directory of Open Access Journals (Sweden)

    Murat Uygun

    2013-01-01

    Full Text Available Poly(methyl methacrylate-co-glycidyl methacrylate (poly(MMA-co-GMA cryogels were synthesized by radical cryopolymerization technique. Then, laccase enzyme was covalently attached to the cryogel and characterized by using swelling studies and SEM and EDX analyses. Kinetic properties and optimum conditions of the immobilized and free laccase were studied and it was found that of the immobilized laccase was lower than that of free laccase. of the immobilized laccase was increased upon immobilization. Optimum pH was found to be 4.0 for each type of laccase, while optimum temperature was shifted to the warmer region after the immobilization. It was also found that thermal stability of the immobilized laccase was higher than that of free laccase. Immobilized laccase could be used for 10 times successive reuse with no significant decrease in its activity. Also, these laccase immobilized cryogels were successfully used for the decolorization of seven different dyes.

  16. Immobilized cells of Candida rugosa possessing fumarase activity

    Energy Technology Data Exchange (ETDEWEB)

    Yang, L.; Zhone, L.

    1980-01-01

    Immobilized cells of C. rugosa that possessed fumarase activity were prepared by different methods; the most active immobilized cells were entrapped in polyacrylamide gels. The effects of pH temperature, and divalent cations on the fumarase activity of both immobilized and native cells were the same. Mn/sup 2 +/, Mg/sup 2 +/, Ca/sup 2 +/, and Fe/sup 2 +/ did not protect the immobilized enzyme against thermal inactivation. The activity of immobilized fumarase remained constant during 91 days of storage of 4-6 degrees. The immobilized cell column was used for the continuous production of L-malic acid from 1M fumarate at 30 degrees and pH 8.5. The immobilized column operated steadily for 2 months. Half life of the immobilized fumarase at 30 degrees was 95 days.

  17. Bed Rest and Immobilization: Risk Factors for Bone Loss

    Science.gov (United States)

    ... Risk Factors for Bone Loss Bed Rest and Immobilization: Risk Factors for Bone Loss Like muscle, bone ... complications of pregnancy; and those who are experiencing immobilization of some part of the body because of ...

  18. Electron transfer kinetics of cytochrome c immobilized on a phenolic terminated thiol self assembled monolayer determined by scanning electrochemical microscopy

    International Nuclear Information System (INIS)

    Alizadeh, Vali; Mousavi, Mir Fazlollah; Mehrgardi, Masoud Ayatollahi; Kazemi, Sayed Habib; Sharghi, Hashem

    2011-01-01

    Highlights: → Preparing a thiolated phenolic self-assembled monolayer surface (SAM). → Application of this SAM to immobilize cytochrome C. → Scanning electrochemical microscopy used for these studies. → Determination of both tunneling electron transfer and bimolecular rate constants between the immobilized protein-substrate and probe. - Abstract: In the present manuscript, the electrochemical behavior of cytochrome c (cyt-c) immobilized onto a phenolic terminated self assembled monolayer (SAM) on a gold electrode is investigated using cyclic voltammetry (CV) and scanning electrochemical microscopy (SECM). The tunneling electron transfer (ET) rate constant between the immobilized protein and the underlying electrode surface, and also the bimolecular ET rate constant between the immobilized protein and a probe has been obtained using approach curves that were obtained by SECM. The approach curves were recorded at different substrate overpotentials in the presence of various concentrations of ferrocyanide as a probe and various surface concentrations of cyt-c; then the standard tunneling ET and bimolecular rate constants are obtained as 3.4 ± 0.3 s -1 and (2.0 ± 0.5) x 10 7 cm 3 mol -1 s -1 , respectively.

  19. Electron transfer kinetics of cytochrome c immobilized on a phenolic terminated thiol self assembled monolayer determined by scanning electrochemical microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Alizadeh, Vali [Department of Chemistry, Tarbiat Modares University, P.O. Box 14115-175, Tehran (Iran, Islamic Republic of); Mousavi, Mir Fazlollah, E-mail: mousavim@modares.ac.ir [Department of Chemistry, Tarbiat Modares University, P.O. Box 14115-175, Tehran (Iran, Islamic Republic of); Mehrgardi, Masoud Ayatollahi [Department of Chemistry, University of Isfahan, Isfahan (Iran, Islamic Republic of); Kazemi, Sayed Habib [Department of Chemistry, Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan (Iran, Islamic Republic of); Sharghi, Hashem [Department of Chemistry, Shiraz University, Shiraz (Iran, Islamic Republic of)

    2011-07-01

    Highlights: > Preparing a thiolated phenolic self-assembled monolayer surface (SAM). > Application of this SAM to immobilize cytochrome C. > Scanning electrochemical microscopy used for these studies. > Determination of both tunneling electron transfer and bimolecular rate constants between the immobilized protein-substrate and probe. - Abstract: In the present manuscript, the electrochemical behavior of cytochrome c (cyt-c) immobilized onto a phenolic terminated self assembled monolayer (SAM) on a gold electrode is investigated using cyclic voltammetry (CV) and scanning electrochemical microscopy (SECM). The tunneling electron transfer (ET) rate constant between the immobilized protein and the underlying electrode surface, and also the bimolecular ET rate constant between the immobilized protein and a probe has been obtained using approach curves that were obtained by SECM. The approach curves were recorded at different substrate overpotentials in the presence of various concentrations of ferrocyanide as a probe and various surface concentrations of cyt-c; then the standard tunneling ET and bimolecular rate constants are obtained as 3.4 {+-} 0.3 s{sup -1} and (2.0 {+-} 0.5) x 10{sup 7} cm{sup 3} mol{sup -1} s{sup -1}, respectively.

  20. Preparation and characterization of two types of covalently immobilized amyloglucosidase

    Directory of Open Access Journals (Sweden)

    ZORAN VUJCIC

    2005-05-01

    Full Text Available Amyloglucosidase from A. niger was covalently immobilized onto poly (GMA-co-EGDMA by the glutaraldehyde and periodate method. The immobilization of amyloglucosidase after periodate oxidation gave a preparate with the highest specific activity reported so far on similar polymers. The obtained immobilized preparates show the same pH optimum, but a higher temperature optimum compared with the soluble enzyme. The kinetic parameters for the hydrolysis of soluble starch by free and both immobilized enzymes were determined.

  1. Immobilization of Isolated Lipase From Moldy Copra (Aspergillus Oryzae)

    OpenAIRE

    Dali, Seniwati; Patong, A. B. D. Rauf; Jalaluddin, M. Noor; Pirman; Hamzah, Baharuddin

    2011-01-01

    Enzyme immobilization is a recovery technique that has been studied in several years, using support as a media to help enzyme dissolutions to the reaction substrate. Immobilization method used in this study was adsorption method, using specific lipase from Aspergillus oryzae. Lipase was partially purified from the culture supernatant of Aspergillus oryzae. Enzyme was immobilized by adsorbed on silica gel. Studies on free and immobilized lipase systems for determination of optimum pH, optimum ...

  2. Short-Term Limb Immobilization Affects Cognitive Motor Processes

    Science.gov (United States)

    Toussaint, Lucette; Meugnot, Aurore

    2013-01-01

    We examined the effects of a brief period of limb immobilization on the cognitive level of action control. A splint placed on the participants' left hand was used as a means of immobilization. We used a hand mental rotation task to investigate the immobilization-induced effects on motor imagery performance (Experiments 1 and 2) and a number mental…

  3. Immobilization of microbial cells: A promising tool for treatment of ...

    African Journals Online (AJOL)

    The review articles on cell immobilization have been published since 1980 and reflect the general interest in this topic. Immobilized microbial cells create opportunities in a wide range of sectors including environmental pollution control. Compared with suspended microorganism technology, cell immobilization shows many ...

  4. Characterization of immobilized post-carbohydrate meal salivary α ...

    African Journals Online (AJOL)

    The effect of experimental parameters like pH, temperature and substrate concentration on the activity of the immobilized post-carbohydrate meal salivary ... of immobilized post-carbohydrate meal salivary α-amylase in this study show that immobilization had no significant effect on the enzyme and compared to kinetic ...

  5. Characteristics of immobilized aminoacylase from Aspergillus oryzae on macroporous copolymers.

    Science.gov (United States)

    He, B L; Jiang, P; Qiu, Y B

    1990-01-01

    Aminoacylase from Aspergillus oryzae was adsorbed on functionallized macroporous copolymers where the enzyme showed excellent catalyzing activity and operation stability. Various factors which effect the activity of the immobilized aminoacylase such as temperature, pH and ionic strength were investigated. The continuous operation of the enzyme immobilized on macroporous copolymers was compared with that of the enzyme immobilized on DEAE-Sephadex.

  6. Immobilization: A Revolution in Traditional Brewing

    Science.gov (United States)

    Virkajärvi, Ilkka; Linko, Matti

    In nature many micro-organisms tend to bind to solid surfaces. This tendency has long been utilized in a number of processes, for example in producing vinegar and acetic acid in bioreactors filled with wood shavings. Acetobacteria are attached to the surface of these shavings. In modern technical language: they are immobilized. Also yeast cells can be immobilized. In the brewing industry this has been the basis for maintaining efficient, continuous fermentation in bioreactors with very high yeast concentrations. The most dramatic change in brewing over recent years has been the replacement of traditional lagering of several weeks by a continuous process in which the residence time is only about 2h. Continuous primary fermentation is used on a commercial scale in New Zealand. In this process, instead of a carrier, yeast is retained in reactors by returning it partly after separation. In many pilot scale experiments the primary fermentation is shortened from about 1week to 1-2days using immobilized yeast reactors. When using certain genetically modified yeast strains no secondary fermentation is needed, and the total fermentation time in immobilized yeast reactors can therefore be shortened to only 2days.

  7. Silica gel matrix immobilized Chlorophyta hydrodictyon africanum ...

    African Journals Online (AJOL)

    Chlorophyta hydrodictyon africanum was immobilized on a silica gel matrix to improve its mechanical properties. The algae-silica gel adsorbent was used for batch sorption studies of a cationic dye, methylene blue (MB). Optimum adsorption was obtained with a dosage of 0.8 g bio sorbent. Results from sorption studies ...

  8. Isomaltulose production using free and immobilized Serratia ...

    African Journals Online (AJOL)

    André

    2016-05-18

    May 18, 2016 ... After 2 h of reaction time in shake flasks, a high production of ... immobilized cells in calcium alginate was studied in a packed bed bioreactor during seven days in a .... cell biomass was obtained from fermentation in a 6.6 L bioreactor .... carbohydrates were analyzed comparing their retention times with.

  9. Immobilization and packaging of recovered tritium

    International Nuclear Information System (INIS)

    Holtslander, W.J.; Miller, J.M.

    1982-09-01

    The evaluation of metal hydrides as a medium for immobilization of tritium is reviewed. The work demonstrated methods of preparation and examined the properties of titanium and zirconium hydride for this application. Methods of packaging the metal hydrides for transportation and recoverable storage of tritium were also examined

  10. Immobilizing Biomolecules Near the Diffraction Limit

    DEFF Research Database (Denmark)

    Skovsen, Esben; Petersen, Maria Teresa Neves; Gennaro, Ane Kold Di

    2009-01-01

    Our group has previously shown that biomolecules containing disulfide bridges in close proximity to aromatic residues can be immobilized, through covalent bonds, onto thiol derivatized surfaces upon UV excitation of the aromatic residue(s). We have also previously shown that our new technology ca...

  11. Enzyme Engineering for In Situ Immobilization.

    Science.gov (United States)

    Rehm, Fabian B H; Chen, Shuxiong; Rehm, Bernd H A

    2016-10-14

    Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.

  12. Metal immobilization in soils using synthetic zeolites

    NARCIS (Netherlands)

    Osté, L.A.; Lexmond, T.M.; Riemsdijk, van W.H.

    2002-01-01

    In situ immobilization of heavy metals in contaminated soils is a technique to improve soil quality. Synthetic zeolites are potentially useful additives to bind heavy metals. This study selected the most effective zeolite in cadmium and zinc binding out of six synthetic zeolites (mordenite-type,

  13. Adsorption of metals by immobilized tannins

    Energy Technology Data Exchange (ETDEWEB)

    Santana, J L; Olivares, S; De La Rosa, D; Martinez, F; Vargas, L M [Centro de Estudios Aplicados al Desarrollo Nuclear (CEADEN), La Habana (Cuba)

    1996-05-01

    Simultaneous adsorption of thorium, europium, cerium, and neodymium by immobilized tannic was studied at different ph values. Tannic materials have excellent ability to adsorb selectively thorium at pH 5. The rest of the elements could be isolated in group at pH 7.

  14. Immobilization of Trichoderma reesei by radiation polymerization

    International Nuclear Information System (INIS)

    Zhou Ruimin; Ma Zueteh; Kaetus, Isao; Kumakura, Minoro

    1993-01-01

    Immobilization of Trichoderma reesei was carried out by radiation polymerization. It was found that the activity of fixed cells increased with increasing surface area of the carrier and was affected by the concentration of monomer tetraethylenglycol dimethacrylate and the shape of the substrate composition and structure of cotton textile fabrics. (author)

  15. Application of radiopolymerization for immobilization of enzymes

    International Nuclear Information System (INIS)

    Higa, O.Z.; Mastro, N.L. del; Castagnet, A.C.G.

    1986-01-01

    Hydrophilic glass-forming monomers were used in an application of irradiation technology for the immobilization of cellulase and cellobiase. Experiments to observe the effect of additives such as silicates and polyethylene glycol in the enzyme entrapment are reported on. In all cases, enzymatic activity was maintained for more than fifteen batch enzyme reactions. (Author) [pt

  16. Plutonium Immobilization Can Loading Preliminary Specifications

    Energy Technology Data Exchange (ETDEWEB)

    Kriikku, E.

    1998-11-25

    This report discusses the Plutonium Immobilization can loading preliminary equipment specifications and includes a process block diagram, process description, equipment list, preliminary equipment specifications, plan and elevation sketches, and some commercial catalogs. This report identifies loading pucks into cans and backfilling cans with helium as the top priority can loading development areas.

  17. Immobilization of Enzymes in Polymer Supports.

    Science.gov (United States)

    Conlon, Hugh D.; Walt, David R.

    1986-01-01

    Two experiments in which an enzyme is immobilized onto a polymeric support are described. The experiments (which also demonstrate two different polymer preparations) involve: (1) entrapping an enzyme in an acrylamide polymer; and (2) reacting the amino groups on the enzyme's (esterase) lysine residues with an activated polymer. (JN)

  18. IN SITU LEAD IMMOBILIZATION BY APATITE

    Science.gov (United States)

    Lead contamination is of environmental concern due to its effect on human health. The purpose of this study was to develop a technology to immobilize Pb in situ in contaminated soils and wastes using apatite. Hydroxyapatite [Ca10(PO4)6(O...

  19. Isomaltulose production using free and immobilized Serratia ...

    African Journals Online (AJOL)

    Isomaltulose is a low cariogenic sweetener used as a substitute for sucrose in the food industry. In this study, isomaltulose production by Serratia plymuthica ATCC 15928 was performed using free and immobilized cells. Response Surface Methodology was employed to evaluate the influence of temperature, wet cell mass ...

  20. Physico-chemical characteristics of immobilized polygalacturonase ...

    African Journals Online (AJOL)

    Polygalacturonase (PG) was isolated from Aspergillus niger (A. niger) (SA6), partially purified, characterized and immobilized by entrapment using calcium alginate. The polygalacturonase showed two bands on sodium dodecyl sulfate polyacryamide gel electrophoresis (SDS-PAGE) suggesting an “endo and exo” ...

  1. Adsorption of metals by immobilized tannins

    International Nuclear Information System (INIS)

    Santana, J.L.; Olivares, S.; De La Rosa, D.; Martinez, F.; Vargas, L.M.

    1996-01-01

    Simultaneous adsorption of thorium, europium, cerium, and neodymium by immobilized tannic was studied at different ph values. Tannic materials have excellent ability to adsorb selectively thorium at pH 5. The rest of the elements could be isolated in group at pH 7

  2. Plasma Treated High-Density Polyethylene (HDPE Medpor Implant Immobilized with rhBMP-2 for Improving the Bone Regeneration

    Directory of Open Access Journals (Sweden)

    Jin-Su Lim

    2014-01-01

    Full Text Available We investigate the bone generation capacity of recombinant human bone morphogenetic protein-2 (rhBMP-2 immobilized Medpor surface through acrylic acid plasma-polymerization. Plasma-polymerization was carried out at a 20 W at an acrylic acid flow rate of 7 sccm for 5 min. The plasma-polymerized Medpor surface showed hydrophilic properties and possessed a high density of carboxyl groups. The rhBMP-2 was immobilized with covalently attached carboxyl groups using 1-ethyl-3-(3-dimethylaminopropyl carbodiimide and N-hydroxysuccinimide. Carboxyl groups and rhBMP-2 immobilization on the Medpor surface were identified by Fourier transform infrared spectroscopy. The activity of Medpor with rhBMP-2 immobilized was examined using an alkaline phosphatase assay on MC3T3-E1 cultured Medpor. These results showed that the rhBMP-2 immobilized Medpor increased the level of MC3T3-E1 cell differentiation. These results demonstrated that plasma surface modification has the potential to immobilize rhBMP-2 on polymer implant such as Medpor and can be used for the binding of bioactive nanomolecules in bone tissue engineering.

  3. Formulation and characterization of lipid-based drug delivery system of raloxifene-microemulsion and self-microemulsifying drug delivery system

    Science.gov (United States)

    Thakkar, Hetal; Nangesh, Jitesh; Parmar, Mayur; Patel, Divyakant

    2011-01-01

    Background: Raloxifene, a second-generation selective estrogen receptor modulator (SERM) used to prevent osteoporosis in postmenopausal women is administered orally in the form of a tablet. The absolute bioavailability of the drug is only 2% because of extensive hepatic first-pass metabolism. Lipid-based formulations are reported to reduce the first-pass metabolism by promoting its lymphatic uptake. Materials and Methods: In the present investigation, microemulsion and Self-Microemulsifying Drug Delivery System (SMEDDS) formulations of Raloxifene were prepared. The prepared formulations were characterized for drug loading, size, transparency, zeta potential, Transmission Electron Microscopy (TEM) and in vitro intestinal permeability. Results: The results indicated that high drug loading, optimum size and desired zeta potential and transparency could be achieved with both SMEDDS and microemulsion. The TEM studies indicated the absence of aggregation with both the systems. The in vitro intestinal permeability results showed that the permeation of the drug from the microemulsion and SMEDDs was significantly higher than that obtained from the drug dispersion and marketed formulation. Conclusion: Lipid based formulations such as microemulsion and Self Microemulsifying drug delivery systems are expected to increase the oral bioavailability as evidenced by the increased intestinal permeation. PMID:21966167

  4. Dissolution of Lipid-Based Matrices in Simulated Gastrointestinal Solutions to Evaluate Their Potential for the Encapsulation of Bioactive Ingredients for Foods.

    Science.gov (United States)

    Raymond, Yves; Champagne, Claude P

    2014-01-01

    The goal of the study was to compare the dissolution of chocolate to other lipid-based matrices suitable for the microencapsulation of bioactive ingredients in simulated gastrointestinal solutions. Particles having approximately 750 μm or 2.5 mm were prepared from the following lipid-based matrices: cocoa butter, fractionated palm kernel oil (FPKO), chocolate, beeswax, carnauba wax, and paraffin. They were added to solutions designed to simulate gastric secretions (GS) or duodenum secretions (DS) at 37°C. Paraffin, carnauba wax, and bees wax did not dissolve in either the GS or DS media. Cocoa butter, FPKO, and chocolate dissolved in the DS medium. Cocoa butter, and to a lesser extent chocolate, also dissolved in the GS medium. With chocolate, dissolution was twice as fast as that with small particles (750 μm) as compared to the larger (2.5 mm) ones. With 750 μm particle sizes, 90% dissolution of chocolate beads was attained after only 60 minutes in the DS medium, while it took 120 minutes for 70% of FPKO beads to dissolve in the same conditions. The data are discussed from the perspective of controlled release in the gastrointestinal tract of encapsulated ingredients (minerals, oils, probiotic bacteria, enzymes, vitamins, and peptides) used in the development of functional foods.

  5. Dissolution of Lipid-Based Matrices in Simulated Gastrointestinal Solutions to Evaluate Their Potential for the Encapsulation of Bioactive Ingredients for Foods

    Directory of Open Access Journals (Sweden)

    Yves Raymond

    2014-01-01

    Full Text Available The goal of the study was to compare the dissolution of chocolate to other lipid-based matrices suitable for the microencapsulation of bioactive ingredients in simulated gastrointestinal solutions. Particles having approximately 750 μm or 2.5 mm were prepared from the following lipid-based matrices: cocoa butter, fractionated palm kernel oil (FPKO, chocolate, beeswax, carnauba wax, and paraffin. They were added to solutions designed to simulate gastric secretions (GS or duodenum secretions (DS at 37°C. Paraffin, carnauba wax, and bees wax did not dissolve in either the GS or DS media. Cocoa butter, FPKO, and chocolate dissolved in the DS medium. Cocoa butter, and to a lesser extent chocolate, also dissolved in the GS medium. With chocolate, dissolution was twice as fast as that with small particles (750 μm as compared to the larger (2.5 mm ones. With 750 μm particle sizes, 90% dissolution of chocolate beads was attained after only 60 minutes in the DS medium, while it took 120 minutes for 70% of FPKO beads to dissolve in the same conditions. The data are discussed from the perspective of controlled release in the gastrointestinal tract of encapsulated ingredients (minerals, oils, probiotic bacteria, enzymes, vitamins, and peptides used in the development of functional foods.

  6. Formulation and characterization of lipid-based drug delivery system of raloxifene-microemulsion and self-microemulsifying drug delivery system

    Directory of Open Access Journals (Sweden)

    Hetal Thakkar

    2011-01-01

    Full Text Available Background : Raloxifene, a second-generation selective estrogen receptor modulator (SERM used to prevent osteoporosis in postmenopausal women is administered orally in the form of a tablet. The absolute bioavailability of the drug is only 2% because of extensive hepatic first-pass metabolism. Lipid-based formulations are reported to reduce the first-pass metabolism by promoting its lymphatic uptake. Materials and Methods : In the present investigation, microemulsion and Self-Microemulsifying Drug Delivery System (SMEDDS formulations of Raloxifene were prepared. The prepared formulations were characterized for drug loading, size, transparency, zeta potential, Transmission Electron Microscopy (TEM and in vitro intestinal permeability. Results : The results indicated that high drug loading, optimum size and desired zeta potential and transparency could be achieved with both SMEDDS and microemulsion. The TEM studies indicated the absence of aggregation with both the systems. The in vitro intestinal permeability results showed that the permeation of the drug from the microemulsion and SMEDDs was significantly higher than that obtained from the drug dispersion and marketed formulation. Conclusion : Lipid based formulations such as microemulsion and Self Microemulsifying drug delivery systems are expected to increase the oral bioavailability as evidenced by the increased intestinal permeation.

  7. Computational Models of the Gastrointestinal Environment. 2. Phase Behavior and Drug Solubilization Capacity of a Type I Lipid-Based Drug Formulation after Digestion.

    Science.gov (United States)

    Birru, Woldeamanuel A; Warren, Dallas B; Han, Sifei; Benameur, Hassan; Porter, Christopher J H; Pouton, Colin W; Chalmers, David K

    2017-03-06

    Lipid-based drug formulations can greatly enhance the bioavailability of poorly water-soluble drugs. Following the oral administration of formulations containing tri- or diglycerides, the digestive processes occurring within the gastrointestinal (GI) tract hydrolyze the glycerides to mixtures of free fatty acids and monoglycerides that are, in turn, solubilized by bile. The behavior of drugs within the resulting colloidal mixtures is currently not well characterized. This work presents matched in vitro experimental and molecular dynamics (MD) theoretical models of the GI microenvironment containing a digested triglyceride-based (Type I) drug formulation. Both the experimental and theoretical models consist of molecular species representing bile (glycodeoxycholic acid), digested triglyceride (1:2 glyceryl-1-monooleate and oleic acid), and water. We have characterized the phase behavior of the physical system using nephelometry, dynamic light scattering, and polarizing light microscopy and compared these measurements to phase behavior observed in multiple MD simulations. Using this model microenvironment, we have investigated the dissolution of the poorly water-soluble drug danazol experimentally using LC-MS and theoretically by MD simulation. The results show how the formulation lipids alter the environment of the GI tract and improve the solubility of danazol. The MD simulations successfully reproduce the experimental results showing the utility of MD in modeling the fate of drugs after digestion of lipid-based formulations within the intestinal lumen.

  8. Minimal impact of an iron-fortified lipid-based nutrient supplement on Hb and iron status

    DEFF Research Database (Denmark)

    James, Philip; Friis, Henrik; Woodd, Susannah

    2015-01-01

    in the intervention arm during the second stage, given from 2 to 6 weeks post-ART. Hb, serum C-reactive protein (CRP), serum ferritin and soluble transferrin receptor (sTfR) were measured at recruitment and 6 weeks post-ART. Multivariable linear regression models were used to assess the impact of the intervention...

  9. Impact of lipid-based drug delivery systems on the transport and uptake of insulin across Caco-2 Cell monolayers

    DEFF Research Database (Denmark)

    Li, Ping; Nielsen, Hanne Mørck; Müllertz, Anette

    2016-01-01

    Self-(nano)-emulsifying drug delivery systems (SNEDDSs) used to deliver peptides and proteins across biological barriers, such as the small intestinal membrane, represents an increasingly interesting field in nanomedicine. Hence, the present study was designed to evaluate the impact of SNEDDS...

  10. Enzyme Immobilization: An Overview on Methods, Support Material, and Applications of Immobilized Enzymes.

    Science.gov (United States)

    Sirisha, V L; Jain, Ankita; Jain, Amita

    Immobilized enzymes can be used in a wide range of processes. In recent years, a variety of new approaches have emerged for the immobilization of enzymes that have greater efficiency and wider usage. During the course of the last two decades, this area has rapidly expanded into a multidisciplinary field. This current study is a comprehensive review of a variety of literature produced on the different enzymes that have been immobilized on various supporting materials. These immobilized enzymes have a wide range of applications. These include applications in the sugar, fish, and wine industries, where they are used for removing organic compounds from waste water. This study also reviews their use in sophisticated biosensors for metabolite control and in situ measurements of environmental pollutants. Immobilized enzymes also find significant application in drug metabolism, biodiesel and antibiotic production, bioremediation, and the food industry. The widespread usage of immobilized enzymes is largely due to the fact that they are cheaper, environment friendly, and much easier to use when compared to equivalent technologies. © 2016 Elsevier Inc. All rights reserved.

  11. Thermal Stabilization of Enzymes Immobilized within Carbon Paste Electrodes.

    Science.gov (United States)

    Wang, J; Liu, J; Cepra, G

    1997-08-01

    In this note we report on the remarkable thermal stabilization of enzymes immobilized in carbon paste electrodes. Amperometric biosensors are shown for the first time to withstand a prolonged high-temperature (>50 °C) stress. Nearly full activity of glucose oxidase is retained over periods of up to 4 months of thermal stress at 60-80 °C. Dramatic improvements in the thermostability are observed for polyphenol oxidase, lactate oxidase, alcohol oxidase, horseradish peroxidase, and amino acid oxidase. Such resistance to heat-induced denaturation is attributed to the conformational rigidity of these biocatalysts within the highly hydrophobic (mineral oil or silicone grease) pasting liquid. While no chemical stabilizer is needed for attaining such protective action, it appears that low humidity (i.e., low water content) is essential for minimizing the protein mobility. Besides their implications for electrochemical biosensors, such observations should lead to a new generation of thermoresistant enzyme reactors based on nonpolar semisolid supports.

  12. Oriented immobilized anti-LDL antibody carrying poly(hydroxyethyl methacrylate) cryogel for cholesterol removal from human plasma

    International Nuclear Information System (INIS)

    Bereli, Nilay; Sener, Guelsu; Yavuz, Handan; Denizli, Adil

    2011-01-01

    Low density lipoprotein (LDL) cholesterol is a major ingredient of the plaque that collects in the coronary arteries and causes coronary heart diseases. Among the methods used for the extracorporeal elimination of LDL from intravasal volume, immunoaffinity technique using anti-LDL antibody as a ligand offers superior selectivity and specificity. Proper orientation of the immobilized antibody is the main issue in immunoaffinity techniques. In this study, anti-human β-lipoprotein antibody (anti-LDL antibody) molecules were immobilized and oriented through protein A onto poly(2-hydroxyethyl methacrylate) (PHEMA) cryogel in order to remove LDL from hypercholesterolemic human plasma. PHEMA cryogel was prepared by free radical polymerization initiated with N,N,N',N'-tetramethylene diamine (TEMED). PHEMA cryogel with a swelling degree of 8.89 g H 2 O/g and 67% macro-porosity was characterized by swelling studies, scanning electron microscope (SEM) and blood compatibility tests. All the clotting times were increased when compared with control plasma. The maximum immobilized anti-LDL antibody amount was 63.2 mg/g in the case of random antibody immobilization and 19.6 mg/g in the case of oriented antibody immobilization (protein A loading was 57.0 mg/g). Random and oriented anti-LDL antibody immobilized PHEMA cryogels adsorbed 111 and 129 mg LDL/g cryogel from hypercholesterolemic human plasma, respectively. Up to 80% of the adsorbed LDL was desorbed. The adsorption-desorption cycle was repeated 6 times using the same cryogel. There was no significant loss of LDL adsorption capacity. - Research highlights: → LDL cholesterol is a risk factor in the development of coronary heart diseases. → Antibodies against LDL are used for the selective extracorporeal removal of LDL. → Protein A is used for the oriented immobilization of anti LDL onto PHEMA cryogel. → PHEMA cryogels are biocompatible, exhibit a low pressure drop, lack diffusion resistance and viscous samples can be

  13. Oriented immobilized anti-LDL antibody carrying poly(hydroxyethyl methacrylate) cryogel for cholesterol removal from human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Bereli, Nilay [Department of Chemistry, Hacettepe University, Beytepe, Ankara (Turkey); Sener, Guelsu [Nanotechnology and Nanomedicine Division, Hacettepe University, Ankara (Turkey); Yavuz, Handan, E-mail: handany@hacettepe.edu.tr [Department of Chemistry, Hacettepe University, Beytepe, Ankara (Turkey); Denizli, Adil [Department of Chemistry, Hacettepe University, Beytepe, Ankara (Turkey)

    2011-07-20

    Low density lipoprotein (LDL) cholesterol is a major ingredient of the plaque that collects in the coronary arteries and causes coronary heart diseases. Among the methods used for the extracorporeal elimination of LDL from intravasal volume, immunoaffinity technique using anti-LDL antibody as a ligand offers superior selectivity and specificity. Proper orientation of the immobilized antibody is the main issue in immunoaffinity techniques. In this study, anti-human {beta}-lipoprotein antibody (anti-LDL antibody) molecules were immobilized and oriented through protein A onto poly(2-hydroxyethyl methacrylate) (PHEMA) cryogel in order to remove LDL from hypercholesterolemic human plasma. PHEMA cryogel was prepared by free radical polymerization initiated with N,N,N',N'-tetramethylene diamine (TEMED). PHEMA cryogel with a swelling degree of 8.89 g H{sub 2}O/g and 67% macro-porosity was characterized by swelling studies, scanning electron microscope (SEM) and blood compatibility tests. All the clotting times were increased when compared with control plasma. The maximum immobilized anti-LDL antibody amount was 63.2 mg/g in the case of random antibody immobilization and 19.6 mg/g in the case of oriented antibody immobilization (protein A loading was 57.0 mg/g). Random and oriented anti-LDL antibody immobilized PHEMA cryogels adsorbed 111 and 129 mg LDL/g cryogel from hypercholesterolemic human plasma, respectively. Up to 80% of the adsorbed LDL was desorbed. The adsorption-desorption cycle was repeated 6 times using the same cryogel. There was no significant loss of LDL adsorption capacity. - Research highlights: {yields} LDL cholesterol is a risk factor in the development of coronary heart diseases. {yields} Antibodies against LDL are used for the selective extracorporeal removal of LDL. {yields} Protein A is used for the oriented immobilization of anti LDL onto PHEMA cryogel. {yields} PHEMA cryogels are biocompatible, exhibit a low pressure drop, lack diffusion

  14. Effects of various spacers between biotin and the phospholipid headgroup on immobilization and sedimentation of biotinylated phospholipid-containing liposomes facilitated by avidin-biotin interactions.

    Science.gov (United States)

    Sakamoto, Yasuhisa; Kikuchi, Koji; Umeda, Kazuaki; Nakanishi, Hiroyuki

    2017-09-01

    Immobilization and sedimentation of liposomes (lipid vesicles) are used in liposome-protein binding assays, facilitated by avidin/streptavidin/NeutrAvidin and biotinylated phospholipid-containing liposomes. Here, we examined the effects of three spacers [six-carbon (X), polyethylene glycol (PEG) 180 (molecular weight 180) and PEG2000 (molecular weight 2,000)] between biotin and the phospholipid headgroup on the immobilization and sedimentation of small unilamellar liposomes/vesicles (SUVs). PEG180 and PEG2000 showed more efficient immobilization of biotinylated SUVs on NeutrAvidin-coated plates than X, but X and PEG180 showed more efficient sedimentation of biotinylated SUVs upon NeutrAvidin addition than PEG2000. Thus, the most appropriate spacers differed between immobilization and sedimentation. A spacer for biotinylated SUVs must be selected according to the particular liposome-protein binding assays examined. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  15. Immobilization of Mortierella vinacea cells by radiation polymerization

    International Nuclear Information System (INIS)

    Kumakura, M.; Kaetsu, I.

    1983-01-01

    Immobilization of Mortierella vinacea cells, which contain active α-galactosidase, by radiation polymerization at low temperatures was studied. The durability of the enzymatic activity of the immobilized cells obtained with hydrophilic monomers was affected by the concentrations of the cells and monomer in which optimum conditions were observed. The enzymatic activity of the immobilized cells obtained with hydrophilic monomers was compared to that of hydrophobic monomers. Michaelis constants of the immobilized cells varied with monomer concentration. The effect of addition of porous solid substances on the immobilization of the cells was studied

  16. Selective Immobilization of Protein Clusters on Polymeric Nanocraters

    NARCIS (Netherlands)

    Valsesia, Andrea; Colpo, Pascal; Meziani, Tarik; Bretagnol, Frederic; Lejeune, Michael; Rossi, François; Bouma, Anita; Garcia Parajo, M.F.

    2006-01-01

    A method for fabricating chemically nanopatterned surfaces based on a combination of colloidal lithography and plasma- enhanced chemical vapor deposition (PECVD) is presented. This method can be applied for the creation of different nanopatterns, and it is in principle not limited in patterning

  17. Development of Lipid-based Nanoparticles for in vivo Targeted Delivery of Imaging Agents into Breast Cancer Cells

    Science.gov (United States)

    2010-04-01

    Fab to recognize pOV8-K b proteins on the surface of live cells, mouse lym phoma cell line EL4 was pulse d with either cognate (pOV8) or irrelevant...120 0.5 3 M FI Time, hour M FI Figure 11. Binding specificity of Pyro-NP-Fab. EL4 cells sensitized with peptide pOV8 ( ) but not with VSV

  18. A fast universal immobilization of immunoglobulin G at 4 °C for the development of array-based immunoassays.

    Directory of Open Access Journals (Sweden)

    Shu-Lin Guo

    Full Text Available To maintain the antibody activity and enhance performance of array-based immunoassays, protein G was used to allow a shorter duration of immunoglobulin G immobilization at 4 °C, with the antibody placed in the appropriate orientation. The multiplexed detection of six pain-related message molecules (PRMMs was used as examples for the development of array-based immunoassays: substance P, calcitonin gene-related peptide, nerve growth factor, brain-derived neurotrophic factor, tumor necrosis factor-α, and β-endorphin. Protein G- and non-protein G-coated slides were tested. Compared to non-protein G immunoassays, protein G shortened the antibody immobilization time at 4 °C from overnight to 2 hours. Only protein G-facilitated immunoassays succeeded in simultaneously detecting all six PRMMs with high specificity. Dose-response curves showed that the limits of detection of the protein G-multiplexed immunoassays for the PRMMs was approximately 164, 167, 120, 60, 80, and 92 pg/ml, respectively. Thus, protein G effectively shortens the duration of antibody immobilization at 4 °C, allowing the use of sensitive array-based immunoassays for the simultaneous detection of PRMMs.

  19. Screening of supports for immobilization of commercial porcine pancreatic lipase

    Directory of Open Access Journals (Sweden)

    Robison Scherer

    2011-12-01

    Full Text Available The aim of this work is to report the performance of different supports for the immobilization of commercial porcine pancreatic lipase. The immobilization tests were carried out in several types of Accurel, activated alumina, kaolin, montmorillonite, ion exchange resins and zeolites. The characterization of the supports showed differences in terms of specific area and morphology. The characteristics of the supports influenced the amount of enzyme adsorbed, yield of immobilization and esterification activity of the resulting immobilized catalyst. The clays KSF and natural and pillared montmorillonites presented potential for use as support for lipase immobilization in terms of yield and esterification activity. Yields of immobilization of 76.32 and 52.01% were achieved for clays KSF and natural montmorillonite, respectively. Esterification activities of 754.03, 595.51, 591.88 and 515.71 U.g-1 were obtained for lipases immobilized in Accurel MP-100, Amberlite XAD-2, mordenite and pillared montmorillonite, respectively.

  20. Immobilization of Isolated Lipase From Moldy Copra (Aspergillus Oryzae

    Directory of Open Access Journals (Sweden)

    Seniwati Dali

    2011-01-01

    Full Text Available Enzyme immobilization is a recovery technique that has been studied in several years, using support as a media to help enzyme dissolutions to the reaction substrate. Immobilization method used in this study was adsorption method, using specific lipase from Aspergillus oryzae. Lipase was partially purified from the culture supernatant of Aspergillus oryzae. Enzyme was immobilized by adsorbed on silica gel. Studies on free and immobilized lipase systems for determination of optimum pH, optimum temperature, thermal stability and reusability were carried out. The results showed that free lipase had optimum pH 8,2 and optimum temperature 35 °C while the immobilized lipase had optimum 8,2 and optimum temperature 45 °C. The thermal stability of the immobilized lipase, relative to that of the free lipase, was markedly increased. The immobilized lipase can be reused for at least six times.

  1. Immobilization of anaerobic thermophilic bacteria for the production of cell-free thermostable. alpha. -amylases and pullulanases

    Energy Technology Data Exchange (ETDEWEB)

    Klingeberg, M [Goettingen Univ. (Germany, F.R.). Inst. fuer Mikrobiologie; Vorlop, K D [Technische Univ. Braunschweig (Germany, F.R.). Inst. fuer Technische Chemie; Antranikian, G [Technische Univ. Hamburg-Harburg, Hamburg (Germany, F. R.). Arbeitsbereich Biotechnologie 1

    1990-08-01

    For the production of cell-free thermostable {alpha}-amylases and pullulanases various anaerobic thermophilic bacteria that belong to the genera Clostridium and Thermoanaerobacter were immobilized in calcium alginate gel beads. The entrapment of bacteria was performed in full was well as in hollow spheres. An optimal limited medium, which avoided bacterial outgrowth, was developed for the cultivation of immobilized organisms at 60deg C using 0.4% starch as substrate. Compared to non-immobilized cells these techniques allowed a significant increase (up to 5.6-fold) in the specific activities of the extracellular enzymes formed. An increase in the productivity of extracellular enzymes was observed after immobilization of bacteria in full spheres. In the case of C. thermosaccharolyticum, for instance, the productivity was raised from 90 units (U)/10{sup 12} cells up to 700 U/10{sup 12} cells. Electrophoretic analysis of the secreted proteins showed that in all cases most of the amylolytic enzymes formed were released into the culture medium. Proteins that had a molecular mass of less than 450 000 daltons could easily diffuse through the gel matrix. Cultivation of immobilized bacteria in semi-continuous and fed-batch cultures was also accompanied by an elevation in the concentration of cell-free enzymes. (orig.).

  2. Effect of Xiaoyaosan Decoction on Learning and Memory Deficit in Rats Induced by Chronic Immobilization Stress

    OpenAIRE

    Meng, Zhen-Zhi; Chen, Jia-Xu; Jiang, You-Ming; Zhang, Han-Ting

    2013-01-01

    Xiaoyaosan (XYS) decoction is a famous prescription which can protect nervous system from stress and treat liver stagnation and spleen deficiency syndrome (LSSDS). In this experiment, we observed the effect of XYS decoction on chronic immobilization stress (CIS) induced learning and memory deficit in rats from behaviors and changes of proteins in hippocampus. We used XYS decoction to treat CIS induced learning and memory deficit in rats with rolipram as positive control, used change of body w...

  3. Continuous throughput and long-term observation of single-molecule FRET without immobilization.

    Science.gov (United States)

    Tyagi, Swati; VanDelinder, Virginia; Banterle, Niccolò; Fuertes, Gustavo; Milles, Sigrid; Agez, Morgane; Lemke, Edward A

    2014-03-01

    We present an automated microfluidic platform that performs multisecond observation of single molecules with millisecond time resolution while bypassing the need for immobilization procedures. With this system, we confine biomolecules to a thin excitation field by reversibly collapsing microchannels to nanochannels. We demonstrate the power of our method by studying a variety of complex nucleic acid and protein systems, including DNA Holliday junctions, nucleosomes and human transglutaminase 2.

  4. Quantification of immobilized Candida antarctica lipase B (CALB) using ICP-AES combined with Bradford method.

    Science.gov (United States)

    Nicolás, Paula; Lassalle, Verónica L; Ferreira, María L

    2017-02-01

    The aim of this manuscript was to study the application of a new method of protein quantification in Candida antarctica lipase B commercial solutions. Error sources associated to the traditional Bradford technique were demonstrated. Eight biocatalysts based on C. antarctica lipase B (CALB) immobilized onto magnetite nanoparticles were used. Magnetite nanoparticles were coated with chitosan (CHIT) and modified with glutaraldehyde (GLUT) and aminopropyltriethoxysilane (APTS). Later, CALB was adsorbed on the modified support. The proposed novel protein quantification method included the determination of sulfur (from protein in CALB solution) by means of Atomic Emission by Inductive Coupling Plasma (AE-ICP). Four different protocols were applied combining AE-ICP and classical Bradford assays, besides Carbon, Hydrogen and Nitrogen (CHN) analysis. The calculated error in protein content using the "classic" Bradford method with bovine serum albumin as standard ranged from 400 to 1200% when protein in CALB solution was quantified. These errors were calculated considering as "true protein content values" the results of the amount of immobilized protein obtained with the improved method. The optimum quantification procedure involved the combination of Bradford method, ICP and CHN analysis. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Immobilization of β-glucosidase onto mesoporous silica support: Physical adsorption and covalent binding of enzyme

    Directory of Open Access Journals (Sweden)

    Ivetić Darjana Ž.

    2014-01-01

    Full Text Available This paper investigates β-glucosidase immobilization onto mesoporous silica support by physical adsorption and covalent binding. The immobilization was carried out onto micro-size silica aggregates with the average pore size of 29 nm. During physical adsorption the highest yield of immobilized β-glucosidase was obtained at initial protein concentration of 0.9 mg ml-1. Addition of NaCl increased 1.7-fold, while Triton X-100 addition decreased 6-fold yield of adsorption in comparison to the one obtained without any addition. Covalently bonded β-glucosidase, via glutaraldehyde previously bonded to silanized silica, had higher yield of immobilized enzyme as well as higher activity and substrate affinity in comparison to the one physically adsorbed. Covalent binding did not considerably changed pH and temperature stability of obtained biocatalyst in range of values that are commonly used in reactions in comparison to unbounded enzyme. Furthermore, covalent binding provided biocatalyst which retained over 70% of its activity after 10 cycles of reuse. [Projekat Ministarstva nauke Republike Srbije, br. III 45021

  6. Design of a papain immobilized antimicrobial food package with curcumin as a crosslinker.

    Directory of Open Access Journals (Sweden)

    Cynthya Maria Manohar

    Full Text Available Contamination of food products by spoilage and pathogenic microorganisms during post process handling is one of the major causes for food spoilage and food borne illnesses. The present green sustainable approach describes the covalent immobilization of papain to LDPE (low density polyethylene, HDPE (high density polyethylene, LLDPE (linear low density polyethylene and PCL (polycaprolactam with curcumin as the photocrosslinker. About 50% of curcumin and 82-92% of papain were successfully immobilized on these polymers. After 30 days, the free enzyme retained 87% of its original activity, while the immobilized enzyme retained more than 90% of its activity on these polymers. Papain crosslinked to LLDPE exhibited the best antibiofilm properties against Acinetobacter sp. KC119137.1 and Staphylococcus aureus NCIM 5021 when compared to the other three polymers, because of the highest amount of enzyme immobilized on this surface. Papain acts by damaging the cell membrane. The enzyme is able to reduce the amount of carbohydrate and protein contents in the biofilms formed by these organisms. Meat wrapped with the modified LDPE and stored at 4°C showed 9 log reduction of these organisms at the end of the seventh day when compared to samples wrapped with the bare polymer. This method of crosslinking can be used on polymers with or without functional groups and can be adopted to bind any type of antimicrobial agent.

  7. Electrochemistry of glucose oxidase immobilized on the carbon nanotube wrapped by polyelectrolyte

    International Nuclear Information System (INIS)

    Wen, Dan; Liu, Ying; Yang, Guocheng; Dong, Shaojun

    2007-01-01

    A more stably dispersing of multi-wall carbon nanotube composite (noted as PDDA-MWNT), which was obtained by wrapping the MWNT with poly(diallydimethylammonium) chloride (PDDA), was used for the immobilization of glucose oxidase (GOD) and its bioelectrochemical studies. The morphologies and structures of the PDDA-MWNT composite were characterized by environment scanning electron microscopy (ESEM) and X-ray photoelectron spectroscopy (XPS). Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry were used to feature the GOD adsorbed onto the electrode modified by PDDA-MWNT composite. The immobilized GOD at the PDDA-MWNT films exhibited a pair of well-defined nearly reversible redox peaks and a fast heterogeneous electron transfer rate with the rate constant (k s ) of 2.76 s -1 . In addition, GOD immobilized in this way retained its bioelectrocatalytic activity for the oxidation of glucose. The method of immobilizing GOD without any additional cross-linking agents presented here is easy and facile, which provides a model for other redox enzymes and proteins

  8. Design of a Papain Immobilized Antimicrobial Food Package with Curcumin as a Crosslinker

    Science.gov (United States)

    Sivakumar, Ponnurengam Malliappan; Doble, Mukesh

    2015-01-01

    Contamination of food products by spoilage and pathogenic microorganisms during post process handling is one of the major causes for food spoilage and food borne illnesses. The present green sustainable approach describes the covalent immobilization of papain to LDPE (low density polyethylene), HDPE (high density polyethylene), LLDPE (linear low density polyethylene) and PCL (polycaprolactam) with curcumin as the photocrosslinker. About 50% of curcumin and 82-92% of papain were successfully immobilized on these polymers. After 30 days, the free enzyme retained 87% of its original activity, while the immobilized enzyme retained more than 90% of its activity on these polymers. Papain crosslinked to LLDPE exhibited the best antibiofilm properties against Acinetobacter sp. KC119137.1 and Staphylococcus aureus NCIM 5021 when compared to the other three polymers, because of the highest amount of enzyme immobilized on this surface. Papain acts by damaging the cell membrane. The enzyme is able to reduce the amount of carbohydrate and protein contents in the biofilms formed by these organisms. Meat wrapped with the modified LDPE and stored at 4°C showed 9 log reduction of these organisms at the end of the seventh day when compared to samples wrapped with the bare polymer. This method of crosslinking can be used on polymers with or without functional groups and can be adopted to bind any type of antimicrobial agent. PMID:25906061

  9. Bioconversion of Airborne Methylamine by Immobilized Recombinant Amine Oxidase from the Thermotolerant Yeast Hansenula polymorpha

    Directory of Open Access Journals (Sweden)

    Sasi Sigawi

    2014-01-01

    Full Text Available Aliphatic amines, including methylamine, are air-pollutants, due to their intensive use in industry and the natural degradation of proteins, amino acids, and other nitrogen-containing compounds in biological samples. It is necessary to develop systems for removal of methylamine from the air, since airborne methylamine has a negative effect on human health. The primary amine oxidase (primary amine : oxygen oxidoreductase (deaminating or amine oxidase, AMO; EC 1.4.3.21, a copper-containing enzyme from the thermotolerant yeast Hansenula polymorpha which was overexpressed in baker’s yeast Saccharomyces cerevisiae, was tested for its ability to oxidize airborne methylamine. A continuous fluidized bed bioreactor (CFBR was designed to enable bioconversion of airborne methylamine by AMO immobilized in calcium alginate (CA beads. The results demonstrated that the bioreactor with immobilized AMO eliminates nearly 97% of the airborne methylamine. However, the enzymatic activity of AMO causes formation of formaldehyde. A two-step bioconversion process was therefore proposed. In the first step, airborne methylamine was fed into a CFBR which contained immobilized AMO. In the second step, the gas flow was passed through another CFBR, with alcohol oxidase from the yeast H. polymorpha immobilized in CA, in order to decompose the formaldehyde formed in the first step. The proposed system provided almost total elimination of the airborne methylamine and the formaldehyde.

  10. Design of a papain immobilized antimicrobial food package with curcumin as a crosslinker.

    Science.gov (United States)

    Manohar, Cynthya Maria; Prabhawathi, Veluchamy; Sivakumar, Ponnurengam Malliappan; Doble, Mukesh

    2015-01-01

    Contamination of food products by spoilage and pathogenic microorganisms during post process handling is one of the major causes for food spoilage and food borne illnesses. The present green sustainable approach describes the covalent immobilization of papain to LDPE (low density polyethylene), HDPE (high density polyethylene), LLDPE (linear low density polyethylene) and PCL (polycaprolactam) with curcumin as the photocrosslinker. About 50% of curcumin and 82-92% of papain were successfully immobilized on these polymers. After 30 days, the free enzyme retained 87% of its original activity, while the immobilized enzyme retained more than 90% of its activity on these polymers. Papain crosslinked to LLDPE exhibited the best antibiofilm properties against Acinetobacter sp. KC119137.1 and Staphylococcus aureus NCIM 5021 when compared to the other three polymers, because of the highest amount of enzyme immobilized on this surface. Papain acts by damaging the cell membrane. The enzyme is able to reduce the amount of carbohydrate and protein contents in the biofilms formed by these organisms. Meat wrapped with the modified LDPE and stored at 4°C showed 9 log reduction of these organisms at the end of the seventh day when compared to samples wrapped with the bare polymer. This method of crosslinking can be used on polymers with or without functional groups and can be adopted to bind any type of antimicrobial agent.

  11. Rapid detection of food pathogens using RNA aptamers-immobilized slide.

    Science.gov (United States)

    Maeng, Jin-Soo; Kim, Namsoo; Kim, Chong-Tai; Han, Seung Ryul; Lee, Young Ju; Lee, Seong-Wook; Lee, Myung-Hyun; Cho, Yong-Jin

    2012-07-01

    The purpose of this study was to develop a simple and rapid detection system for foodborne bacteria, which consisted of an optical microscope and its slide chip with artificial antibodies, or RNA aptamers. From an RNA pool, three each RNA aptamers were built by the method of SELEX (systematic evolution of ligands by exponential enrichment) for components of cell wall, LPS (lipopolysaccharide) from E. coli O157:H7, teichoic acid from Staphylococcus aureus and a cell membrane protein of OmpC from Salmonella typhimurium, respectively. These aptamers were hybridized with thiol-conjugated 16 dT-linker molecules in order to be immobilized on silver surface which was, in advance, fabricated on glass slide, using a spin-coating method. To confirm that each aptamers retained its specific binding activities to their antigenic live bacteria, microscopic view of bound cells immobilized on silver film were observed. Furthermore, we observed the fluorescence-emitting bacteria-aptamer complex immobilized on silver film after adding RNA aptamers hybridized with fluorophore, FAM-conjugated 16 dT-linker molecules. As a result, the RNA aptamers-immobilized slide system developed in this study was a useful new tool to rapidly monitor individual food pathogens.

  12. Electrochemical and spectroelectrochemical characterization of different mesoporous TiO2 film electrodes for the immobilization of Cytochrome c

    Science.gov (United States)

    Katsiaounis, Stavros; Tiflidis, Christina; Tsekoura, Christina; Topoglidis, Emmanuel

    2018-03-01

    In this work three different mesoporous TiO2 film electrodes were prepared and used for the immobilization of Cytochrome c (Cyt-c). Films prepared via a standard sol-gel route (SG-films) were compared with commercially available benchmark nanotitania materials, namely P25 Degussa (P25-films) and Dyesol nanopaste (Dyesol films). Their properties, film deposition characteristics and their abilities to adsorb protein molecules in a stable and functional way were examined. We investigated whether it is possible, rather than preparing TiO2 films using multistep, lengthy and not always reproducible sol-gel procedures, to use commercially available nanotitania materials and produce reproducible films faster that exhibit all the properties that make TiO2 films ideal for protein immobilization. Although these materials are formulated primarily for dye-sensitized solar cell applications, in this study we found out that protein immobilization is facile and remarkably stable on all of them. We also investigated their electrochemical properties by using cyclic voltammetry and spectroelectrochemistry and found out that not only direct reduction of Fe(III)-heme to Fe(II)-heme of immobilized Cyt-c was possible on all films but that the adsorbed protein remained electroactive.

  13. Molecular characteristics of an immobilization antigen gene of the fish-parasitic protozoan Ichthyophthirius multifiliis strain ARS-6

    Science.gov (United States)

    Ichthyophthirius multifiliis, a ciliated protozoan parasite of fish, expresses surface antigens (i-antigens), which react with host antibodies that render them immobile. The nucleotide sequence of an i-antigen gene of Ichthyophthirius multifiliis strain ARS-6 was deduced. The predicted protein of 47...

  14. Comparison of different transition metal ions for immobilized metal affinity chromatography of selenoprotein P from human plasma

    DEFF Research Database (Denmark)

    Sidenius, U; Farver, O; Jøns, O

    1999-01-01

    Cu2+, Ni2+, Zn2+, Co2+ and Cd2+ were evaluated in metal ion affinity chromatography for enrichment of selenoprotein P, and immobilized Co2+ affinity chromatography was found to be the most selective chromatographic method. The chromatography was performed by fast protein liquid chromatography...

  15. Technologies for immobilization and disposal of tritium

    International Nuclear Information System (INIS)

    Coppari, N.R.

    1996-01-01

    This study was done within a program one of whose objectives was to know the state of the technology development for tritium separation in the moderator circuit at HWR and to define the possible technologies to be applied to the Argentine nuclear power plants. Within this framework the strategies adopted by each country and the available technologies for a safe disposal of tritium, not only in its gaseous state tritium but also as tritiated water were analyzed. It is considered that if the selected separation method is such that the tritium is in its gaseous state, the hydride formation for long periods of immobilization should be studied. whereas if it were triated water immobilization should be studied to choose the technology between cementation and drying agents, in both cases the final disposal site will have to be selected. (author). 8 refs

  16. Fluctuation correlation models for receptor immobilization

    Science.gov (United States)

    Fourcade, B.

    2017-12-01

    Nanoscale dynamics with cycles of receptor diffusion and immobilization by cell-external-or-internal factors is a key process in living cell adhesion phenomena at the origin of a plethora of signal transduction pathways. Motivated by modern correlation microscopy approaches, the receptor correlation functions in physical models based on diffusion-influenced reaction is studied. Using analytical and stochastic modeling, this paper focuses on the hybrid regime where diffusion and reaction are not truly separable. The time receptor autocorrelation functions are shown to be indexed by different time scales and their asymptotic expansions are given. Stochastic simulations show that this analysis can be extended to situations with a small number of molecules. It is also demonstrated that this analysis applies when receptor immobilization is coupled to environmental noise.

  17. Immobilization of IFR salt wastes in mortar

    International Nuclear Information System (INIS)

    Fisher, D.F.; Johnson, T.R.

    1988-01-01

    Portland cement-base mortars are being considered for immobilizing chloride salt wastes from the fuel cycle of an integral fast reactor (IFR). The IFR is a sodium-cooled fast reactor with metal fuel. It has a close-coupled fuel cycle in which fission products are separated from the actinides in an electrochemical cell operating at 500 degrees C. This cell has a cadmium anode and a liquid salt electrolyte. The salt will be a low-melting mixture of alkaline and alkaline earth chlorides. This paper discusses one method being considered for immobilizing this treated salt, to disperse it in a portland cement-base motar, which would then be sealed in corrosion-resistant containers. For this application, the grout must be sufficiently fluid that it can be pumped into canisters where it will solidify into a strong, leach-resistant material

  18. Capture and immobilization of krypton-85

    International Nuclear Information System (INIS)

    Whitmell, D.S.; Geens, L.; Penzhorn, R.D.; Smith, M.J.S.

    1985-01-01

    It may become necessary to contain the krypton-85 released from nuclear fuel during reprocessing in order to reduce the exposure to the local population and the radioactive background throughout the world. A brief description is given of studies being carried out in the Indirect Action Programme. The separation of krypton from other off-gases by cryogenic distillation in the presence of oxygen is being studied at SCK/CEN Mol, together with the behavior of ozone in the distillation column. Two processes for the immobilization of krypton in solid forms have been successfully developed and demonstrated. At KfK Karlsruhe, krypton is encapsulated in vitrified zeolites; at AERE Harwell, krypton is immobilized within a metallic matrix. These processes offer excellent gas retention and either could be adopted for a reprocessing plant

  19. Weight and height prediction of immobilized patients

    OpenAIRE

    Rabito,Estela Iraci; Vannucchi,Gabriela Bergamini; Suen,Vivian Marques Miguel; Castilho Neto,Laércio Lopes; Marchini,Júlio Sérgio

    2006-01-01

    OBJECTIVE: To confirm the adequacy of the formula suggested in the literature and/or to develop appropriate equations for the Brazilian population of immobilized patients based on simple anthropometric measurements. METHODS: Hospitalized patients were submitted to anthropometry and methods to estimate weight and height of bedridden patients were developed by multiple linear regression. RESULTS: Three hundred sixty eight persons were evaluated at two hospital centers and five weight-predicting...

  20. Immobilized yeast in bioreactor for alcohol fermentation

    International Nuclear Information System (INIS)

    Handy, M.K.; Kim, K.

    1986-01-01

    Mutant of Saccharomyces cerevisiae was developed using a Co-60 source. Cells were immobilized onto sterile, channeled alumina beads and packed into bioreactor column under controlled temperature. Feedstocks containing substrate and nutrients were fed into the bioreactor at specific rates. Beads with greatest porosity and surface area produced the most ethanol. Factors affecting ethanol productivity included: temperature, pH, flow rate, nutrients and substrate in the feedstock

  1. Immobilization of radioactive waste in glass matrices

    International Nuclear Information System (INIS)

    Wicks, G.G.

    1978-01-01

    A promising process for long-term management of high-level radioactive waste is to immobilize the waste in a borosilicate glass matrix. Among the most important criteria characterizing the integrity of the large-scale glass-waste forms are that they possess good chemical stability (including low leachability), thermal stability, mechanical integrity, and high radiation stability. Fulfillment of these criteria ensures the maximum margin of safety of glass-waste products, following solidification, handling, transportation, and long-term storage

  2. Optimization of Immobilization of Nanodiamonds on Graphene

    International Nuclear Information System (INIS)

    Pille, A; Lange, S; Utt, K; Eltermann, M

    2015-01-01

    We report using simple dip-coating method to cover the surface of graphene with nanodiamonds for future optical detection of defects on graphene. Most important part of the immobilization process is the pre-functionalization of both, nanodiamond and graphene surfaces to obtain the selectiveness of the method. This work focuses on an example of using electrostatic attraction to confine nanodiamonds to graphene. Raman spectroscopy, microluminescence imaging and scanning electron microscopy were applied to characterize obtained samples. (paper)

  3. Process arrangement options for Defense waste immobilization

    International Nuclear Information System (INIS)

    1980-02-01

    Current plans are to immobilize the SRP high-level liquid wastes in a high integrity form. Borosilicate glass was selected in 1977 as the reference waste form and a mjaor effort is currently underway to develop the required technology. A large new facility, referred to as the Defense Waste Processing Facility (DWPF) is being designed to carry out this mission, with project authorization targeted for 1982 and plant startup in 1989. However, a number of other process arrangements or manufacturing strategies, including staging the major elements of the project or using existing SRP facilities for some functions, have been suggested in lieu of building the reference DWPF. This study assesses these various options and compares them on a technical and cost basis with the DWPF. Eleven different manufacturing options for SRP defense waste solidification were examined in detail. These cases are: (1) vitrification of acid waste at current generation rate; (2) vitrification of current rate acid waste and caustic sludge; (3 and 4) vitrification of the sludge portion of neutralized waste; (5) decontamination of salt cake and storage of concentrated cesium and strontium for later immobilization; (6) processing waste in a facility with lower capacity than the DWPF; (7) processing waste in a combination of existing and new facilities; (8) waste immobilization in H Canyon; (9) vitrification of both sludge and salt; (10) DWPF with onsite storage; (11) deferred authorization of DWPF

  4. Comparative physical-chemical characterization of encapsulated lipid-based isotretinoin products assessed by particle size distribution and thermal behavior analyses

    Energy Technology Data Exchange (ETDEWEB)

    Guimaraes, Carla Aiolfi, E-mail: carlaaiolfi@usp.br [Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, SP 05508-000 (Brazil); Menaa, Farid [Department of Dermatology, School of Medicine Wuerzburg, Wuerzburg 97080 (Germany); Fluorotronics, Inc., 1425 Russ Bvld, San Diego Technology Incubator, San Diego, CA 92101 (United States); Menaa, Bouzid, E-mail: bouzid.menaa@gmail.com [Fluorotronics, Inc., 1425 Russ Bvld, San Diego Technology Incubator, San Diego, CA 92101 (United States); Quenca-Guillen, Joyce S. [Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, SP 05508-000 (Brazil); Matos, Jivaldo do Rosario [Department of Fundamental Chemistry, Institute of Chemistry, University of Sao Paulo, Sao Paulo, SP 05508-000 (Brazil); Mercuri, Lucildes Pita [Department of Exact and Earth Sciences, Federal University of Sao Paulo, Diadema, SP 09972-270 (Brazil); Braz, Andre Borges [Department of Engineering of Mines and Oil, Polytechnical School, University of Sao Paulo, SP 05508-900 (Brazil); Rossetti, Fabia Cristina [Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP 14015-120 (Brazil); Kedor-Hackmann, Erika Rosa Maria; Santoro, Maria Ines Rocha Miritello [Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, SP 05508-000 (Brazil)

    2010-06-10

    Isotretinoin is the drug of choice for the management of severe recalcitrant nodular acne. Nevertheless, some of its physical-chemical properties are still poorly known. Hence, the aim of our study consisted to comparatively evaluate the particle size distribution (PSD) and characterize the thermal behavior of the three encapsulated isotretinoin products in oil suspension (one reference and two generics) commercialized in Brazil. Here, we show that the PSD, estimated by laser diffraction and by polarized light microscopy, differed between the generics and the reference product. However, the thermal behavior of the three products, determined by thermogravimetry (TGA), differential thermal (DTA) analyses and differential scanning calorimetry (DSC), displayed no significant changes and were more thermostable than the isotretinoin standard used as internal control. Thus, our study suggests that PSD analyses in isotretinoin lipid-based formulations should be routinely performed in order to improve their quality and bioavailability.

  5. Acceptability of new formulations of corn-soy blends and lipid-based nutrient supplements in Province du Passoré, Burkina Faso

    DEFF Research Database (Denmark)

    Iuel-Brockdorff, Ann-Sophie Julie D; Dræbel, Tania; Fabiansen, Christian

    2015-01-01

    The objective of this study was to evaluate the acceptability of new formulations of six corn-soy blended flours (CSB) and six lipid-based nutrient supplements (LNS) with different quantities of milk and qualities of soy to be used for the treatment of moderate acute malnutrition (MAM). Furthermore......, we wanted to explore the acceptability of foods currently used for the prevention and treatment of malnutrition in Burkina Faso to identify possible barriers that could affect the acceptability of the new formulations of supplementary foods. The study was carried out prior to a randomized controlled...... trial evaluating the effectiveness of these new formulations. The study involved an observed test-meal and a three-day take-home ration of the experimental food supplements to 6-30-months-old healthy children, followed by questionnaire-based interviews about the acceptability of these supplements...

  6. Lipid-Based Formulations Can Enable the Model Poorly Water-Soluble Weakly Basic Drug Cinnarizine to Precipitate in an Amorphous-Salt Form during in Vitro Digestion

    DEFF Research Database (Denmark)

    Khan, Jamal; Rades, Thomas; Boyd, Ben J

    2016-01-01

    The tendency for poorly water-soluble weakly basic drugs to precipitate in a noncrystalline form during the in vitro digestion of lipid-based formulations (LBFs) was linked to an ionic interaction between drug and fatty acid molecules produced upon lipid digestion. Cinnarizine was chosen as a model...... from the starting free base crystalline material to the hydrochloride salt, thus supporting the case that ionic interactions between weak bases and fatty acid molecules during digestion are responsible for producing amorphous-salts upon precipitation. The conclusion has wide implications...... weakly basic drug and was dissolved in a medium-chain (MC) LBF, which was subject to in vitro lipolysis experiments at various pH levels above and below the reported pKa value of cinnarizine (7.47). The solid-state form of the precipitated drug was analyzed using X-ray diffraction (XRD), Fourier...

  7. The effectiveness of immobilization during prostate irradiation

    International Nuclear Information System (INIS)

    Bentel, Gunilla C.; Marks, Lawrence B.; Sherouse, George W.; Spencer, David P.; Anscher, Mitchell S.

    1995-01-01

    Purpose: To evaluate the effect of a hemibody foam cradle on the reproducibility of patient setup during external beam radiation treatment of prostate cancer. Methods and Materials: Between January 1992 and April 1993, 74 patients received external beam radiation treatment to the prostate ± nodes, generally with a four-field box technique. Forty-four of the 74 patients had a custom-made hemibody foam cast used in an attempt to improve setup accuracy. A review of the routine weekly port films was performed following the completion of therapy to determine the reproducibility of patient setup in all 74 patients. The physician's request of an isocenter shift was used as an indicator of reproducibility. Neither the treating technologists nor the physicians knew at the time the films were taken that the port films would be reviewed for setup reproducibility at a later date. The results were compared between the patients treated with (44) and without (30) an immobilization device. Results: In the 44 immobilized patients, 213 routine checks of the isocenter were performed during the 7-week course of radiation therapy. In 17.4% of these instances (37 out of 213), an isocenter shift was requested. This rate is compared to 23.1% (30 out of 130) in the 30 patients who did not have the immobilization device (p < 0.2). There was a statistically significant reduction in isocenter shifts requested in the anterior to posterior direction in the patients who were immobilized, 5.1% (9 out of 175) vs. 12.6% (13 out of 103) (p < 0.05, two tailed chi-square test). There was no significant improvement in the reproducibility of isocenter placement in the cephalad to caudal or right to left directions. Conclusions: This custom-made hemibody foam cradle appears to improve the reproducibility of patient setup during the 7-week course of fractionated external beam irradiation for patients with adenocarcinoma of the prostate. This type of immobilization device is now routinely used in our

  8. Enzyme immobilization and biocatalysis of polysiloxanes

    Science.gov (United States)

    Poojari, Yadagiri

    Lipases have been proven to be versatile and efficient biocatalysts which can be used in a broad variety of esterification, transesterification, and ester hydrolysis reactions. Due to the high chemo-, regio-, and stereo-selectivity and the mild conditions of lipase-catalyzed reactions, the vast potential of these biocatalysts for use in industrial applications has been increasingly recognized. Polysiloxanes (silicones) are well known for their unique physico-chemical properties and can be prepared in the form of fluids, elastomers, gels and resins for a wide variety of applications. However, the enzymatic synthesis of silicone polyesters and copolymers is largely unexplored. In the present investigations, an immobilized Candida antarctica lipase B (CALB) on macroporous acrylic resin beads (Novozym-435 RTM) has been successfully employed as a catalyst to synthesize silicone polyesters and copolymers under mild reaction conditions. The silicone aliphatic polyesters and the poly(dimethylsiloxane)--poly(ethylene glycol) (PDMS-PEG) copolymers were synthesized in the bulk (without using a solvent), while the silicone aromatic polyesters, the silicone aromatic polyamides and the poly(epsilon-caprolactone)--poly(dimethylsiloxane)--poly(epsilon-caprolactone) (PCL-PDMS-PCL) triblock copolymers were synthesized in toluene. The synthesized silicone polyesters and copolymers were characterized by Gel Permeation Chromatography (GPC), Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD). This dissertation also describes a methodology for physical immobilization of the enzyme pepsin from Porcine stomach mucosa in silicone elastomers utilizing condensation-cure room temperature vulcanization (RTV) of silanol-terminated poly(dimethylsiloxane) (PDMS). The activity and the stability of free pepsin and pepsin immobilized in silicone elastomers were studied with respect to p

  9. Lipase immobilization and production of fatty acid methyl esters from canola oil using immobilized lipase

    International Nuclear Information System (INIS)

    Yuecel, Yasin; Demir, Cevdet; Dizge, Nadir; Keskinler, Buelent

    2011-01-01

    Lipase enzyme from Aspergillus oryzae (EC 3.1.1.3) was immobilized onto a micro porous polymeric matrix which contains aldehyde functional groups and methyl esters of long chain fatty acids (biodiesel) were synthesized by transesterification of crude canola oil using immobilized lipase. Micro porous polymeric matrix was synthesized from styrene-divinylbenzene (STY-DVB) copolymers by using high internal phase emulsion technique and two different lipases, Lipozyme TL-100L ® and Novozym 388 ® , were used for immobilization by both physical adsorption and covalent attachment. Biodiesel production was carried out with semi-continuous operation. Methanol was added into the reactor by three successive additions of 1:4 M equivalent of methanol to avoid enzyme inhibition. The transesterification reaction conditions were as follows: oil/alcohol molar ratio 1:4; temperature 40 o C and total reaction time 6 h. Lipozyme TL-100L ® lipase provided the highest yield of fatty acid methyl esters as 92%. Operational stability was determined with immobilized lipase and it indicated that a small enzyme deactivation occurred after used repeatedly for 10 consecutive batches with each of 24 h. Since the process is yet effective and enzyme does not leak out from the polymer, the method can be proposed for industrial applications. -- Research highlights: → Lipozyme TL-100L and Novozym 388 were immobilized onto micro porous polymeric matrix by both physical adsorption and covalent linking. → Immobilized enzymes were used for synthesis of fatty acid methyl esters by transesterification of canola oil and methanol using semi-continuous operation system. → According to chromatographic analysis, Lipase Lipozyme TL-100L resulted in the highest yield of methyl ester as 92%.

  10. Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: Characterization and application for enzymatic inhibition assays

    International Nuclear Information System (INIS)

    Zhu, Yuan-Ting; Ren, Xiao-Yun; Liu, Yi-Ming; Wei, Ying; Qing, Lin-Sen; Liao, Xun

    2014-01-01

    Using carboxyl functionalized silica-coated magnetic nanoparticles (MNPs) as carrier, a novel immobilized porcine pancreatic lipase (PPL) was prepared through the 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. Transmission electron microscopic images showed that the synthesized nanoparticles (Fe 3 O 4 –SiO 2 ) possessed three dimensional core–shell structures with an average diameter of ∼ 20 nm. The effective enzyme immobilization onto the nanocomposite was confirmed by atomic force microscopic (AFM) analysis. Results from Fourier-transform infrared spectroscopy (FT-IR), Bradford protein assay, and thermo-gravimetric analysis (TGA) indicated that PPL was covalently attached to the surface of magnetic nanoparticles with a PPL immobilization yield of 50 mg enzyme/g MNPs. Vibrating sample magnetometer (VSM) analysis revealed that the MNPs-PPL nanocomposite had a high saturation magnetization of 42.25 emu·g −1 . The properties of the immobilized PPL were investigated in comparison with the free enzyme counterpart. Enzymatic activity, reusability, thermo-stability, and storage stability of the immobilized PPL were found significantly superior to those of the free one. The K m and the V max values (0.02 mM, 6.40 U·mg −1 enzyme) indicated the enhanced activity of the immobilized PPL compared to those of the free enzyme (0.29 mM, 3.16 U·mg −1 enzyme). Furthermore, at an elevated temperature of 70 °C, immobilized PPL retained 60% of its initial activity. The PPL-MNPs nanocomposite was applied in the enzyme inhibition assays using orlistat, and two natural products isolated from oolong tea (i.e., EGCG and EGC) as the test compounds. - Highlights: • Porcine pancreatic lipase was firstly covalently immobilized onto carboxylated MNPs. • Immobilized porcine pancreatic lipase (PPL) was characterized by various techniques. • MNPs-PPL showed higher activity, reusability, and thermo-stability than

  11. Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: Characterization and application for enzymatic inhibition assays

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Yuan-Ting [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Ren, Xiao-Yun [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041 (China); Liu, Yi-Ming [Department of Chemistry and Biochemistry, Jackson State University, 1400 Lynch St., Jackson, MS 39217 (United States); Wei, Ying [Changzhi Medical College, Changzhi 046000 (China); Qing, Lin-Sen [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041 (China); Liao, Xun, E-mail: liaoxun@cib.ac.cn [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041 (China)

    2014-05-01

    Using carboxyl functionalized silica-coated magnetic nanoparticles (MNPs) as carrier, a novel immobilized porcine pancreatic lipase (PPL) was prepared through the 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. Transmission electron microscopic images showed that the synthesized nanoparticles (Fe{sub 3}O{sub 4}–SiO{sub 2}) possessed three dimensional core–shell structures with an average diameter of ∼ 20 nm. The effective enzyme immobilization onto the nanocomposite was confirmed by atomic force microscopic (AFM) analysis. Results from Fourier-transform infrared spectroscopy (FT-IR), Bradford protein assay, and thermo-gravimetric analysis (TGA) indicated that PPL was covalently attached to the surface of magnetic nanoparticles with a PPL immobilization yield of 50 mg enzyme/g MNPs. Vibrating sample magnetometer (VSM) analysis revealed that the MNPs-PPL nanocomposite had a high saturation magnetization of 42.25 emu·g{sup −1}. The properties of the immobilized PPL were investigated in comparison with the free enzyme counterpart. Enzymatic activity, reusability, thermo-stability, and storage stability of the immobilized PPL were found significantly superior to those of the free one. The K{sub m} and the V{sub max} values (0.02 mM, 6.40 U·mg{sup −1} enzyme) indicated the enhanced activity of the immobilized PPL compared to those of the free enzyme (0.29 mM, 3.16 U·mg{sup −1} enzyme). Furthermore, at an elevated temperature of 70 °C, immobilized PPL retained 60% of its initial activity. The PPL-MNPs nanocomposite was applied in the enzyme inhibition assays using orlistat, and two natural products isolated from oolong tea (i.e., EGCG and EGC) as the test compounds. - Highlights: • Porcine pancreatic lipase was firstly covalently immobilized onto carboxylated MNPs. • Immobilized porcine pancreatic lipase (PPL) was characterized by various techniques. • MNPs-PPL showed higher activity

  12. Collagen Type III and VI Turnover in Response to Long-Term Immobilization.

    Directory of Open Access Journals (Sweden)

    Shu Sun

    Full Text Available Muscle mass and function are perturbed by immobilization and remobilization. When muscle mass changes, the quality and quantity of the extracellular matrix protein, particularly the collagens, change with it. In this study, we investigated the temporal profile of three peptide biomarkers derived from turnover of collagen type III and type VI in a long-term immobilization and remobilization study. We also compared individual biomarker levels with Lean body Mass (LBM and changes therein, hypothesizing that these biomarkers would be biomarkers of the remodeling processes associated with immobilization and/or remobilization.In the Berlin bed rest study, 20 young men were recruited and randomly assigned to 8-week's strict bed rest with or without resistive vibration exercise countermeasure. We measured three neo-epitope ELISA kits in the serum samples of this study: Pro-C3, measured the synthesis of collagen type III; Pro-C6, measured the synthesis of collagen type VI; and C6M measured the degradation of collagen type VI induced by MMP-2 and MMP-9 cleavage.Pro-C3 and Pro-C6 biomarkers are up-regulated with both immobilization and remobilization, whereas C6M is hardly affected at all. We found that Pro-C3 and C6M levels are related to LBM at baseline and that high levels of Pro-C6 are associated with smaller changes in muscle mass during both immobilization and remobilization.The Pro-C3 and-C6 biomarkers change likely reflect remodeling changes in response to unloading or reloading, whereas C6M does not appear to respond to unloading. Pro-C3 and C6M levels correlate with LBM at baseline, while Pro-C6 is related to the anabolic and catabolic responses to unloading and reloading.

  13. Preparation of immobilized growing cells and enzymatic hydrolysis of sawdust

    International Nuclear Information System (INIS)

    Kumakura, M.; Kaetsu, I.

    1984-01-01

    Trichoderma reesei cells were immobilized by radiation polymerization using porous materials such as non-woven material and sawdust, and the enzymatic hydrolysis of sawdust with the enzyme solution from the immobilized growing cells was studied. The filter paper activity, which shows the magnitude of cellulase production in the immobilized cells, was comparable with that in the intact cells. The filter paper activity was affected by addition concentration of monomer and porous materials. The cells in the immobilized cells grew to be adhered on the surface of the fibrous polymers. Sawdust, which was pretreated by irradiation technique, was effectively hydrolyzed with the enzyme solution resulting from the culture of the immobilized cells, in which the glucose yield increased increasing the culture time of the immobilized cells. (author)

  14. Ceramic membrane microfilter as an immobilized enzyme reactor.

    Science.gov (United States)

    Harrington, T J; Gainer, J L; Kirwan, D J

    1992-10-01

    This study investigated the use of a ceramic microfilter as an immobilized enzyme reactor. In this type of reactor, the substrate solution permeates the ceramic membrane and reacts with an enzyme that has been immobilized within its porous interior. The objective of this study was to examine the effect of permeation rate on the observed kinetic parameters for the immobilized enzyme in order to assess possible mass transfer influences or shear effects. Kinetic parameters were found to be independent of flow rate for immobilized penicillinase and lactate dehydrogenase. Therefore, neither mass transfer nor shear effects were observed for enzymes immobilized within the ceramic membrane. Both the residence time and the conversion in the microfilter reactor could be controlled simply by regulating the transmembrane pressure drop. This study suggests that a ceramic microfilter reactor can be a desirable alternative to a packed bed of porous particles, especially when an immobilized enzyme has high activity and a low Michaelis constant.

  15. Treatment and immobilization of intermediate level radioactive wastes

    International Nuclear Information System (INIS)

    Lerch, R.E.; Greenhalgh, W.O.; Partridge, J.A.; Richardson, G.L.

    1977-01-01

    This paper discusses a new program underway to develop and demonstrate treatment and immobilization technologies for intermediate level wastes (ILW) generated in the nuclear fuel cycle. Initial work has defined the sources, quantities and types of wastes which comprise ILW. Laboratory studies are underway to define treatment technologies for liquid ILW which contains volatile contaminants and to define immobilization parameters for the residues resulting from treatment of ILW. Immobilization agents initially being evaluated for the various residues include cement, urea-formaldehyde, and bitumen although other immobilization agents will be studied. The program also includes development of acceptable test procedures for the final immobilized products as well as development of proposed criteria for storage, transportation, and disposal of the immobilized ILW. 20 figures, 10 tables

  16. Insulin action in human thighs after one-legged immobilization

    DEFF Research Database (Denmark)

    Richter, Erik; Kiens, Bente; Mizuno, M.

    1989-01-01

    Insulin action was assessed in thighs of five healthy young males who had one knee immobilized for 7 days by a splint. The splint was not worn in bed. Subjects also used crutches to prevent weight bearing of the immobilized leg. Immobilization decreased the activity of citrate synthase and 3-OH......-acyl-CoA-dehydrogenase in the vastus lateralis muscle by 9 and 14%, respectively, and thigh volume by 5%. After 7 days of immobilization, a two-step euglycemic hyperinsulinemic clamp procedure combined with arterial and bilateral femoral venous catheterization was performed. Insulin action on glucose uptake and tyrosine release...... of the thighs at mean plasma insulin concentrations of 67 (clamp step I) and 447 microU/ml (clamp step II) was decreased by immobilization, whereas immobilization did not affect insulin action on thigh exchange of free fatty acids, glycerol, O2, or potassium. Before and during the clamp step I, lactate release...

  17. Hierarchically Nanoporous Bioactive Glasses for High Efficiency Immobilization of Enzymes

    DEFF Research Database (Denmark)

    He, W.; Min, D.D.; Zhang, X.D.

    2014-01-01

    Bioactive glasses with hierarchical nanoporosity and structures have been heavily involved in immobilization of enzymes. Because of meticulous design and ingenious hierarchical nanostructuration of porosities from yeast cell biotemplates, hierarchically nanostructured porous bioactive glasses can...... and products of catalytic reactions can freely diffuse through open mesopores (2–40 nm). The formation mechanism of hierarchically structured porous bioactive glasses, the immobilization mechanism of enzyme and the catalysis mechanism of immobilized enzyme are then discussed. The novel nanostructure...

  18. Catalytical Properties of Free and Immobilized Aspergillus niger Tannase

    OpenAIRE

    Abril Flores-Maltos; Luis V. Rodríguez-Durán; Jacqueline Renovato; Juan C. Contreras; Raúl Rodríguez; Cristóbal N. Aguilar

    2011-01-01

    A fungal tannase was produced, recovered, and immobilized by entrapment in calcium alginate beads. Catalytical properties of the immobilized enzyme were compared with those of the free one. Tannase was produced intracellularly by the xerophilic fungus Aspergillus niger GH1 in a submerged fermentation system. Enzyme was recovered by cell disruption and the crude extract was partially purified. The catalytical properties of free and immobilized tannase were evaluated using tannic acid and methy...

  19. Platform for immobilization and observation of subcellular processes

    Science.gov (United States)

    McKnight, Timothy E.; Kalluri, Udaya C.; Melechko, Anatoli V.

    2014-08-26

    A method of immobilizing matter for imaging that includes providing an array of nanofibers and directing matter to the array of the nanofibers. The matter is immobilized when contacting at least three nanofibers of the array of nanofibers simultaneously. Adjacent nanofibers in the array of nanofibers may be separated by a pitch as great as 100 microns. The immobilized matter on the array of nanofibers may then be imaged. In some examples, the matter may be cell matter, such as protoplasts.

  20. The effects of using immobilizer made of the polyurethane foam

    International Nuclear Information System (INIS)

    Kim, Yong Hwan; Chung, Sang Hwa; Kim, Hwa Young; Lim, Shin Taek; Oh, Sae Dong

    1992-01-01

    The immogilzer for control the patient movement during the radiation beam on was made of the polyurethane foam. The time consumption is abut 8 minutes for completely making the immobilizer. In this experimental study, the effects of using the individual immobilizer have shown that the patients have had always a same position with comfortable and high reproducibilities. Furthermore, it has shown the time for patient setup was decreased by using the individual patient immobilizer.

  1. Bioelectrochemistry of non-covalent immobilized alcohol dehydrogenase on oxidized diamond nanoparticles.

    Science.gov (United States)

    Nicolau, Eduardo; Méndez, Jessica; Fonseca, José J; Griebenow, Kai; Cabrera, Carlos R

    2012-06-01

    Diamond nanoparticles are considered a biocompatible material mainly due to their non-cytotoxicity and remarkable cellular uptake. Model proteins such as cytochrome c and lysozyme have been physically adsorbed onto diamond nanoparticles, proving it to be a suitable surface for high protein loading. Herein, we explore the non-covalent immobilization of the redox enzyme alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae (E.C.1.1.1.1) onto oxidized diamond nanoparticles for bioelectrochemical applications. Diamond nanoparticles were first oxidized and physically characterized by X-ray diffraction (XRD), FT-IR and TEM. Langmuir isotherms were constructed to investigate the ADH adsorption onto the diamond nanoparticles as a function of pH. It was found that a higher packing density is achieved at the isoelectric point of the enzyme. Moreover, the relative activity of the immobilized enzyme on diamond nanoparticles was addressed under optimum pH conditions able to retain up to 70% of its initial activity. Thereafter, an ethanol bioelectrochemical cell was constructed by employing the immobilized alcohol dehydrogenase onto diamond nanoparticles, this being able to provide a current increment of 72% when compared to the blank solution. The results of this investigation suggest that this technology may be useful for the construction of alcohol biosensors or biofuel cells in the near future. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Upregulation of interleukin-1β/transforming growth factor-β1 and hypoxia relate to molecular mechanisms underlying immobilization-induced muscle contracture.

    Science.gov (United States)

    Honda, Yuichiro; Sakamoto, Junya; Nakano, Jiro; Kataoka, Hideki; Sasabe, Ryo; Goto, Kyo; Tanaka, Miho; Origuchi, Tomoki; Yoshimura, Toshiro; Okita, Minoru

    2015-09-01

    In this study we investigated the molecular mechanism underlying muscle contracture in rats. The rats were divided into immobilization and control groups, and soleus muscles of the right and left sides were selected for analyses. The levels of CD11b and α-SMA protein, IL-1β, and TGF-β1 mRNA, and type I and III collagen protein and mRNA were significantly greater in the immobilization group than in the control group at all time-points. HIF-1α mRNA levels were significantly higher in the immobilization group at 4 weeks. Moreover, HIF-1α, α-SMA, and type I collagen levels were significantly higher at 4 weeks than at 1 and 2 weeks in the immobilization group. In the early stages of immobilization, upregulation of IL-1β/TGF-β1 via macrophages may promote fibroblast differentiation that could affect muscle contracture. The soleus muscle became hypoxic in the later stages of immobilization, suggesting that hypoxia influences the progression of muscle contracture. © 2014 Wiley Periodicals, Inc.

  3. Multifunctional Cationic Lipid-Based Nanoparticles Facilitate Endosomal Escape and Reduction-Triggered Cytosolic siRNA Release

    Science.gov (United States)

    Gujrati, Maneesh; Malamas, Anthony; Shin, Tesia; Jin, Erlei; Sun, Lulu; Lu, Zheng-Rong

    2015-01-01

    Small interfering RNA (siRNA) has garnered much attention in recent years as a promising avenue for cancer gene therapy due to its ability to silence disease-related genes. Effective gene silencing is contingent upon the delivery of siRNA into the cytosol of target cells and requires the implementation of delivery systems possessing multiple functionalities to overcome delivery barriers. The present work explores the multifunctional properties and biological activity of a recently developed cationic lipid carrier, (1-aminoethyl)iminobis[N-(oleicylcysteinyl-1-amino-ethyl)propionamide]) (ECO). The physicochemical properties and biological activity of ECO/siRNA nanoparticles were assessed over a range of N/P ratios to optimize the formulation. Potent and sustained luciferase silencing in a U87 glioblastoma cell line was observed, even in the presence of serum proteins. ECO/siRNA nanoparticles exhibited pH-dependent membrane disruption at pH levels corresponding to various stages of the intracellular trafficking pathway. It was found that disulfide linkages created during nanoparticle formation enhanced the protection of siRNA from degradation and facilitated site-specific siRNA release in the cytosol by glutathione-mediated reduction. Confocal microscopy confirmed that ECO/siRNA nanoparticles readily escaped from late endosomes prior to cytosolic release of the siRNA cargo. These results demonstrate that the rationally designed multifunctionality of ECO/siRNA nanoparticles is critical for intracellular siRNA delivery and the continuing development of safe and effective delivery systems. PMID:25020033

  4. Increase in stability of cellulase immobilized on functionalized magnetic nanospheres

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wenjuan [Department of Machine Intelligence and Systems Engineering, Faculty of Systems Engineering, Akita Prefectural University, Akita 015-0055 (Japan); Qiu, Jianhui, E-mail: qiu@akita-pu.ac.jp [Department of Machine Intelligence and Systems Engineering, Faculty of Systems Engineering, Akita Prefectural University, Akita 015-0055 (Japan); Feng, Huixia [College of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou 730050 (China); Zang, Limin; Sakai, Eiichi [Department of Machine Intelligence and Systems Engineering, Faculty of Systems Engineering, Akita Prefectural University, Akita 015-0055 (Japan)

    2015-02-01

    Functionalized magnetic nanospheres were prepared by co-condensation of tetraethylorthosilicate with three different amino-silanes: 3-(2-aminoethylamino propyl)-triethoxysilane (AEAPTES), 3-(2-aminoethylamino propyl)-trimethoxysilane (AEAPTMES) and 3-aminopropyltriethoxysilane (APTES). Then three functionalized magnetic nanospheres were used as supports for immobilization of cellulase. The three functionalized magnetic nanospheres with core–shell morphologies exhibited higher capacity for cellulase immobilization than unfunctionalized magnetic nanospheres. The increasing of surface charge of functionalized magnetic nanospheres leads to an enhancement of the capacity of cellulase immobilization. Particularly, AEAPTMES with methoxy groups was favored to be hydrolyzed and grafted on unfunctionalized magnetic nanospheres than the others. AEAPTMES functionalized magnetic nanospheres with the highest zeta potential (29 mV) exhibited 87% activity recovery and the maximum amount of immobilized cellulase was 112 mg/g support at concentration of initial cellulase of 8 mg/mL. Immobilized cellulase on AEAPTMES functionalized magnetic nanospheres had higher temperature stability and broader pH stability than other immobilized cellulases and free cellulase. In particular, it can be used in about 40 °C, demonstrating the potential of biofuel production using this immobilized cellulase. - Highlights: • Three Amino-silane modified magnetic nanospheres were prepared. • Cellulase immobilized AEAPTMES functionalized magnetic nanospheres had higher temperature stability and broader pH stability than free cellulase. • The potential of biofuel production using this immobilized cellulase.

  5. Immobilization of thorium over fibroin by polyacrylonitrile (PAN)

    International Nuclear Information System (INIS)

    Aslani, M.A.A.; Akyil, S.; Eral, M.

    1997-01-01

    This report describes a process for immobilization of thorium over fibroin, which was used as a bio-adsorbant, by polyacrylonitrile. The amounts of thorium in aqueous solutions which may be leached in various aqueous ambients were detected by a spectrophotometer. The results show that polyacrylonitrile processes are feasible to immobilize spent fibroins. The leachability of the materials immobilized with polyacrylonitrile can meet the requirements of storage and final disposal. The leachability of thorium ions from immobilized spent fibroin was rather low for 8 months

  6. Application of magnetic nanoparticles in smart enzyme immobilization.

    Science.gov (United States)

    Vaghari, Hamideh; Jafarizadeh-Malmiri, Hoda; Mohammadlou, Mojgan; Berenjian, Aydin; Anarjan, Navideh; Jafari, Nahideh; Nasiri, Shahin

    2016-02-01

    Immobilization of enzymes enhances their properties for efficient utilization in industrial processes. Magnetic nanoparticles, due to their high surface area, large surface-to-volume ratio and easy separation under external magnetic fields, are highly valued. Significant progress has been made to develop new catalytic systems that are immobilized onto magnetic nanocarriers. This review provides an overview of recent developments in enzyme immobilization and stabilization protocols using this technology. The current applications of immobilized enzymes based on magnetic nanoparticles are summarized and future growth prospects are discussed. Recommendations are also given for areas of future research.

  7. Immobilization of yeast cells by radiation-induced polymerization

    International Nuclear Information System (INIS)

    Fujimura, T.; Kaetsu, I.

    1982-01-01

    Radiation-induced polymerization method was applied to the immobilization of yeast cells. The effects of irradiation, cooling and monomer, which are neccessary for polymerization, were recovered completely by subsequent aerobical incubation of yeast cells. The ethanol productive in immobilized yeast cells increased with the increase of aerobical incubation period. The growth of yeast cells in immobilized yeast cells was indicated. The maximum ethanol productivity in immobilized yeast cell system was around three times as much as that in free yeast cell system. (orig.)

  8. Management of immobilization and its complication for elderly.

    Science.gov (United States)

    Laksmi, Purwita W; Harimurti, Kuntjoro; Setiati, Siti; Soejono, Czeresna H; Aries, Wanarani; Roosheroe, Arya Govinda

    2008-10-01

    Increased life expectancy have an effect on the rising percentage of elderly population in Indonesia and health problem associated with the elderly, particularly immobilization. Immobilization may cause various complications, especially when it has been overlooked without any appropriate and proper medical care in keeping with the procedures. High incidence of immobilization in elderly and the life-threatening complication call for an agreement on management of immobilization and its complication. Management of immobilization needs interdisciplinary team-work cooperation, the patients and their family. The management may be commenced through a complete geriatric review, formulating functional goals and constructing therapeutic plan. Various medical conditions and external factors that may act as risk factors of immobilization as well as drugs intake that may exaggerate the immobilization should be evaluated and optimally managed. Any complication due to immobilization and other concomitant disease/condition should be recognized and managed comprehensively in order to reduce morbidity and mortality. Management of immobilization and its complications include pharmacological and non-pharmacological treatment, i.e. various mobility exercises, utilization of ambulatory device and supporting appliance for assisting patients in stand-up position, as well as the management of urinary voiding and defecation.

  9. Increase in stability of cellulase immobilized on functionalized magnetic nanospheres

    International Nuclear Information System (INIS)

    Zhang, Wenjuan; Qiu, Jianhui; Feng, Huixia; Zang, Limin; Sakai, Eiichi

    2015-01-01

    Functionalized magnetic nanospheres were prepared by co-condensation of tetraethylorthosilicate with three different amino-silanes: 3-(2-aminoethylamino propyl)-triethoxysilane (AEAPTES), 3-(2-aminoethylamino propyl)-trimethoxysilane (AEAPTMES) and 3-aminopropyltriethoxysilane (APTES). Then three functionalized magnetic nanospheres were used as supports for immobilization of cellulase. The three functionalized magnetic nanospheres with core–shell morphologies exhibited higher capacity for cellulase immobilization than unfunctionalized magnetic nanospheres. The increasing of surface charge of functionalized magnetic nanospheres leads to an enhancement of the capacity of cellulase immobilization. Particularly, AEAPTMES with methoxy groups was favored to be hydrolyzed and grafted on unfunctionalized magnetic nanospheres than the others. AEAPTMES functionalized magnetic nanospheres with the highest zeta potential (29 mV) exhibited 87% activity recovery and the maximum amount of immobilized cellulase was 112 mg/g support at concentration of initial cellulase of 8 mg/mL. Immobilized cellulase on AEAPTMES functionalized magnetic nanospheres had higher temperature stability and broader pH stability than other immobilized cellulases and free cellulase. In particular, it can be used in about 40 °C, demonstrating the potential of biofuel production using this immobilized cellulase. - Highlights: • Three Amino-silane modified magnetic nanospheres were prepared. • Cellulase immobilized AEAPTMES functionalized magnetic nanospheres had higher temperature stability and broader pH stability than free cellulase. • The potential of biofuel production using this immobilized cellulase

  10. Immobilization and therapeutic passive stretching generate thickening and increase the expression of laminin and dystrophin in skeletal muscle

    International Nuclear Information System (INIS)

    Cação-Benedini, L.O.; Ribeiro, P.G.; Prado, C.M.; Chesca, D.L.; Mattiello-Sverzut, A.C.

    2014-01-01

    Extracellular matrix and costamere proteins transmit the concentric, isometric, and eccentric forces produced by active muscle contraction. The expression of these proteins after application of passive tension stimuli to muscle remains unknown. This study investigated the expression of laminin and dystrophin in the soleus muscle of rats immobilized with the right ankle in plantar flexion for 10 days and subsequent remobilization, either by isolated free movement in a cage or associated with passive stretching for up to 10 days. The intensity of the macrophage response was also evaluated. One hundred and twenty-eight female Wistar rats were divided into 8 groups: free for 10 days; immobilized for 10 days; immobilized/free for 1, 3, or 10 days; or immobilized/stretched/free for 1, 3, or 10 days. After the experimental procedures, muscle tissue was processed for immunofluorescence (dystrophin/laminin/CD68) and Western blot analysis (dystrophin/laminin). Immobilization increased the expression of dystrophin and laminin but did not alter the number of macrophages in the muscle. In the stretched muscle groups, there was an increase in dystrophin and the number of macrophages after 3 days compared with the other groups; dystrophin showed a discontinuous labeling pattern, and laminin was found in the intracellular space. The amount of laminin was increased in the muscles treated by immobilization followed by free movement for 10 days. In the initial stages of postimmobilization (1 and 3 days), an exacerbated macrophage response and an increase of dystrophin suggested that the therapeutic stretching technique induced additional stress in the muscle fibers and costameres

  11. Immobilization and therapeutic passive stretching generate thickening and increase the expression of laminin and dystrophin in skeletal muscle

    Energy Technology Data Exchange (ETDEWEB)

    Cação-Benedini, L.O.; Ribeiro, P.G. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Medicina e Reabilitação do Aparelho Locomotor, Departamento de Biomecânica, Ribeirão Preto, SP, Brasil, Departamento de Biomecânica, Medicina e Reabilitação do Aparelho Locomotor, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Prado, C.M.; Chesca, D.L. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Patologia, Ribeirão Preto, SP, Brasil, Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Mattiello-Sverzut, A.C. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Medicina e Reabilitação do Aparelho Locomotor, Departamento de Biomecânica, Ribeirão Preto, SP, Brasil, Departamento de Biomecânica, Medicina e Reabilitação do Aparelho Locomotor, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2014-05-09

    Extracellular matrix and costamere proteins transmit the concentric, isometric, and eccentric forces produced by active muscle contraction. The expression of these proteins after application of passive tension stimuli to muscle remains unknown. This study investigated the expression of laminin and dystrophin in the soleus muscle of rats immobilized with the right ankle in plantar flexion for 10 days and subsequent remobilization, either by isolated free movement in a cage or associated with passive stretching for up to 10 days. The intensity of the macrophage response was also evaluated. One hundred and twenty-eight female Wistar rats were divided into 8 groups: free for 10 days; immobilized for 10 days; immobilized/free for 1, 3, or 10 days; or immobilized/stretched/free for 1, 3, or 10 days. After the experimental procedures, muscle tissue was processed for immunofluorescence (dystrophin/laminin/CD68) and Western blot analysis (dystrophin/laminin). Immobilization increased the expression of dystrophin and laminin but did not alter the number of macrophages in the muscle. In the stretched muscle groups, there was an increase in dystrophin and the number of macrophages after 3 days compared with the other groups; dystrophin showed a discontinuous labeling pattern, and laminin was found in the intracellular space. The amount of laminin was increased in the muscles treated by immobilization followed by free movement for 10 days. In the initial stages of postimmobilization (1 and 3 days), an exacerbated macrophage response and an increase of dystrophin suggested that the therapeutic stretching technique induced additional stress in the muscle fibers and costameres.

  12. Optimizing Immobilized Enzyme Performance in Cell-Free Environments to Produce Liquid Fuels

    Energy Technology Data Exchange (ETDEWEB)

    Belfort, Georges [Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering; Grimaldi, Joseph J. [Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering

    2015-01-27

    Limitations on biofuel production using cell culture (Escherichia coli, Clostridium, Saccharomyces cerevisiae, brown microalgae, blue-green algae and others) include low product (alcohol) concentrations (≤0.2 vol%) due to feedback inhibition, instability of cells, and lack of economical product recovery processes. To overcome these challenges, an alternate simplified biofuel production scheme was tested based on a cell-free immobilized enzyme system. Using this cell free system, we were able to obtain about 2.6 times higher concentrations of iso-butanol using our non-optimized system as compared with live cell systems. This process involved two steps: (i) converts acid to aldehyde using keto-acid decarboxylase (KdcA), and (ii) produces alcohol from aldehyde using alcohol dehydrogenase (ADH) with a cofactor (NADH) conversion from inexpensive formate using a third enzyme, formate dehydrogenase (FDH). To increase stability and conversion efficiency with easy separations, the first two enzymes were immobilized onto methacrylate resin. Fusion proteins of labile KdcA (fKdcA) were expressed to stabilize the covalently immobilized KdcA. Covalently immobilized ADH exhibited long-term stability and efficient conversion of aldehyde to alcohol over multiple batch cycles without fusions. High conversion rates and low protein leaching were achieved by covalent immobilization of enzymes on methacrylate resin. The complete reaction scheme was demonstrated by immobilizing both ADH and fKdcA and using FDH free in solution. The new system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.5 % (v/v). Further increases in titer will require continuous removal of the isobutanol using our novel brush membrane system that exhibits a 1.5 fold increase in the separation factor of isobutanol from water versus that obtained for commercial silicone rubber membranes. These bio-inspired brush membranes are based on the

  13. Evaluation of inorganic matrixes as supports for immobilization of microbial lipase

    Directory of Open Access Journals (Sweden)

    Castro H.F.

    2000-01-01

    Full Text Available Candida rugosa was immobilized by physical adsorption on several inorganic supports using hexane as coupling medium. The enzymatic activities of the different derivatives were determined by both hydrolysis of olive oil and esterification of n-butanol with butyric acid. The results were compared to previous data obtained by using a controlled porous silica matrix. The goal was to contribute in searching inexpensive supports for optimum lipase performance. All supports examined exhibited good properties for binding the enzyme lipase. Zirconium phosphate was the best support, giving the highest percentage of protein fixation (86% and the highest retention of lipase activity after immobilization (34%. The operational stability performance for niobium oxide derivative was improved by previously activated the support with silane and glutaraldehyde. Thermal stabilities were also examined by thermal gravimetric analysis (TG.

  14. Synthesis of tripodal catecholates and their immobilization on zinc oxide nanoparticles

    Directory of Open Access Journals (Sweden)

    Franziska Klitsche

    2015-05-01

    Full Text Available A common approach to generate tailored materials and nanoparticles (NPs is the formation of molecular monolayers by chemisorption of bifunctional anchor molecules. This approach depends critically on the choice of a suitable anchor group. Recently, bifunctional catecholates, inspired by mussel-adhesive proteins (MAPs and bacterial siderophores, have received considerable interest as anchor groups for biomedically relevant metal surfaces and nanoparticles. We report here the synthesis of new tripodal catecholates as multivalent anchor molecules for immobilization on metal surfaces and nanoparticles. The tripodal catecholates have been conjugated to various effector molecules such as PEG, a sulfobetaine and an adamantyl group. The potential of these conjugates has been demonstrated with the immobilization of tripodal catecholates on ZnO NPs. The results confirmed a high loading of tripodal PEG-catecholates on the particles and the formation of stable PEG layers in aqueous solution.

  15. Electric fields control the orientation of peptides irreversibly immobilized on radical-functionalized surfaces.

    Science.gov (United States)

    Martin, Lewis J; Akhavan, Behnam; Bilek, Marcela M M

    2018-01-24

    Surface functionalization of an implantable device with bioactive molecules can overcome adverse biological responses by promoting specific local tissue integration. Bioactive peptides have advantages over larger protein molecules due to their robustness and sterilizability. Their relatively small size presents opportunities to control the peptide orientation on approach to a surface to achieve favourable presentation of bioactive motifs. Here we demonstrate control of the orientation of surface-bound peptides by tuning electric fields at the surface during immobilization. Guided by computational simulations, a peptide with a linear conformation in solution is designed. Electric fields are used to control the peptide approach towards a radical-functionalized surface. Spontaneous, irreversible immobilization is achieved when the peptide makes contact with the surface. Our findings show that control of both peptide orientation and surface concentration is achieved simply by varying the solution pH or by applying an electric field as delivered by a small battery.

  16. Arsenic mobilization and immobilization in paddy soils

    Science.gov (United States)

    Kappler, A.; Hohmann, C.; Zhu, Y. G.; Morin, G.

    2010-05-01

    Arsenic is oftentimes of geogenic origin and in many cases bound to iron(III) minerals. Iron(III)-reducing bacteria can harvest energy by coupling the oxidation of organic or inorganic electron donors to the reduction of Fe(III). This process leads either to dissolution of Fe(III)-containing minerals and thus to a release of the arsenic into the environment or to secondary Fe-mineral formation and immobilisation of arsenic. Additionally, aerobic and anaerobic iron(II)-oxidizing bacteria have the potential to co-precipitate or sorb arsenic during iron(II) oxidation at neutral pH that is usually followed by iron(III) mineral precipitation. We are currently investigating arsenic immobilization by Fe(III)-reducing bacteria and arsenic co-precipitation and immobilization by anaerobic iron(II)-oxidizing bacteria in batch, microcosm and rice pot experiments. Co-precipitation batch experiments with pure cultures of nitrate-dependent Fe(II)-oxidizing bacteria are used to quantify the amount of arsenic that can be immobilized during microbial iron mineral precipitation, to identify the minerals formed and to analyze the arsenic binding environment in the precipitates. Microcosm and rice pot experiments are set-up with arsenic-contaminated rice paddy soil. The microorganisms (either the native microbial population or the soil amended with the nitrate-dependent iron(II)-oxidizing Acidovorax sp. strain BoFeN1) are stimulated either with iron(II), nitrate, or oxygen. Dissolved and solid-phase arsenic and iron are quantified. Iron and arsenic speciation and redox state in batch and microcosm experiments are determined by LC-ICP-MS and synchrotron-based methods (EXAFS, XANES).

  17. Spectroscopic Evidence of Uranium Immobilization in Acidic ...

    Science.gov (United States)

    Biogeochemistry of uranium in wetlands plays important roles in U immobilization in storage ponds of U mining and processing facilities but has not been well understood. The objective of this work was to study molecular mechanisms responsible for high U retention by Savannah River Site (SRS) wetland sediments under varying redox and acidic (pH = 2.6-5.8) conditions using U L3-edge X-ray absorption spectroscopy. Uranium in the SRS wetland sediments existed primarily as U(VI) bonded as a bidentate to carboxylic sites (U-C bond distance at ~2.88 Å), rather than phenolic or other sites of natural organic matter (NOM). In microcosms simulating the SRS wetland process, U immobilization on roots was 2 orders of magnitude higher than on the adjacent brown or more distant white sands in which U was U(VI). Uranium on the roots were both U(IV) and U(VI), which were bonded as a bidentate to carbon, but the U(VI) may also form a U phosphate mineral. After 140 days of air exposure, all U(IV) was reoxidized to U(VI) but remained as a bidentate bonding to carbon. This study demonstrated NOM and plant roots can highly immobilize U(VI) in the SRS acidic sediments, which has significant implication on the long-term stewardship of U-contaminated wetlands. There were several former U processing facilities at the Savannah River Site (SRS), Aiken, SC. As a result of their operations, uranium has entered the surrounding environments. For example, approximately 45,000 kg o

  18. Plutonium Immobilization Program cold pour tests

    International Nuclear Information System (INIS)

    Hovis, G.L.; Stokes, M.W.; Smith, M.E.; Wong, J.W.

    1999-01-01

    The Plutonium Immobilization Program (PIP) is a joint venture between the Savannah River Site, Lawrence Livermore National Laboratory, Argonne National Laboratory, and Pacific Northwest National Laboratory to carry out the disposition of excess weapons-grade plutonium. This program uses the can-in-canister (CIC) approach. CIC involves encapsulating plutonium in ceramic forms (or pucks), placing the pucks in sealed stainless steel cans, placing the cans in long cylindrical magazines, latching the magazines to racks inside Defense Waste Processing Facility (DWPF) canisters, and filling the DWPF canisters with high-level waste glass. This process puts the plutonium in a stable form and makes it attractive for reuse. At present, the DWPF pours glass into empty canisters. In the CIC approach, the addition of a stainless steel rack, magazines, cans, and ceramic pucks to the canisters introduces a new set of design and operational challenges: All of the hardware installed in the canisters must maintain structural integrity at elevated (molten-glass) temperatures. This suggests that a robust design is needed. However, the amount of material added to the DWPF canister must be minimized to prevent premature glass cooling and excessive voiding caused by a large internal thermal mass. High metal temperatures, minimizing thermal mass, and glass flow paths are examples of the types of technical considerations of the equipment design process. To determine the effectiveness of the design in terms of structural integrity and glass-flow characteristics, full-scale testing will be conducted. A cold (nonradioactive) pour test program is planned to assist in the development and verification of a baseline design for the immobilization canister to be used in the PIP process. The baseline design resulting from the cold pour test program and CIC equipment development program will provide input to Title 1 design for second-stage immobilization. The cold pour tests will be conducted in two

  19. Remote handling in the Plutonium Immobilization Project: Plutonium conversion and first stage immobilization

    International Nuclear Information System (INIS)

    Brault, J.R.

    2000-01-01

    Since the break up of the Soviet Union at the end of the Cold War, the United States and Russia have been negotiating ways to reduce their nuclear stockpiles. Economics is one of the reasons behind this, but another important reason is safeguarding these materials from unstable organizations and countries. With the downsizing of the nuclear stockpiles, large quantities of plutonium are being declared excess and must be safely disposed of. The Savannah River Site (SRS) has been selected as the site where the immobilization facility will be located. Conceptual design and process development commenced in 1998. SRS will immobilize excess plutonium in a ceramic waste form and encapsulate it in vitrified high level waste in the Defense Waste Processing Facility (DWPF) canister. These canisters will then be interred in the national repository at Yucca Mountain, New Mexico. The facility is divided into three distinct operating areas: Plutonium Conversion, First Stage Immobilization, and Second Stage Immobilization. This paper will discuss the first two operations

  20. Immobilized humic substances and immobilized aggregates of humic substances as sorbent for solid phase extraction.

    Science.gov (United States)

    Erny, Guillaume L; Gonçalves, Bruna M; Esteves, Valdemar I

    2013-09-06

    In this work, humic substances (HS) immobilized, as a thin layer or as aggregates, on silica gel were tested as material for solid phase extraction. Some triazines (simazine, atrazine, therbutylazine, atrazine-desethyl-desisopropyl-2-hydroxy, ametryn and terbutryn), have been selected as test analytes due to their environmental importance and to span a large range of solubility and octanol/water partition coefficient (logP). The sorbent was obtained immobilizing a thin layer of HS via physisorption on a pre-coated silica gel with a cationic polymer (polybrene). While the sorbent could be used as it is, it was demonstrated that additional HS could be immobilized, via weak interactions, to form stable humic aggregates. However, while a higher quantity of HS could be immobilized, no significant differences were observed in the sorption parameters. This sorbent have been tested for solid phase extraction to concentrate triazines from aqueous matrixes. The sorbent demonstrated performances equivalent to commercial alternatives as a concentration factor between 50 and 200, depending on the type of triazines, was obtained. Moreover the low cost and the high flow rate of sample through the column allowed using high quantity of sorbent. The analytical procedure was tested with different matrixes including tap water, river water and estuarine water. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Phosphopeptide enrichment by immobilized metal affinity chromatography

    DEFF Research Database (Denmark)

    Thingholm, Tine E.; Larsen, Martin R.

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively...... charged metal ions such as Fe3+, Ga3+, Al3+, Zr4+, and Ti4+ has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from...

  2. Immobilized enzyme studies in a microscale bioreactor.

    Science.gov (United States)

    Jones, Francis; Forrest, Scott; Palmer, Jim; Lu, Zonghuan; Elmore, John; Elmore, Bill B

    2004-01-01

    Novel microreactors with immobilized enzymes were fabricated using both silicon and polymer-based microfabrication techniques. The effectiveness of these reactors was examined along with their behavior over time. Urease enzyme was successfully incorporated into microchannels of a polymeric matrix of polydimethylsiloxane and through layer-bylayer self-assembly techniques onto silicon. The fabricated microchannels had cross-sectional dimensions ranging from tens to hundreds of micrometers in width and height. The experimental results for continuous-flow microreactors are reported for the conversion of urea to ammonia by urease enzyme. Urea conversions of >90% were observed.

  3. Tritium immobilization and packaging using metal hydrides

    International Nuclear Information System (INIS)

    Holtslander, W.J.; Yaraskavitch, J.M.

    1981-04-01

    Tritium recovered from CANDU heavy water reactors will have to be packaged and stored in a safe manner. Tritium will be recovered in the elemental form, T 2 . Metal tritides are effective compounds in which to immobilize the tritium as a stable non-reactive solid with a high tritium capacity. The technology necessary to prepare hydrides of suitable metals, such as titanium and zirconium, have been developed and the properties of the prepared materials evaluated. Conceptual designs of packages for containing metal tritides suitable for transportation and long-term storage have been made and initial testing started. (author)

  4. Immobilization-associated osteoporosis in primates

    Science.gov (United States)

    Young, D. R.; Niklowitz, W. J.; Brown, R. J.; Jee, W. S. S.

    1986-01-01

    Osteopenic changes in the tibial compact bone of fifteen adult male monkeys immobilized for up to 7 months are examined histologically. Osteonal formation in the proximal tibia is analyzed. The analysis reveals the loss of haversian bone in the proximal tibia, increased activation with excessive depth of penetration of osteoclastic activity, rapid bone loss, and resorption cavities of irregular size and orientation. Osteonal formation following reambulation is examined; the recovery of cortical is a repair and rejuvenation process characterized by refilling of resorption cavities and remodeling activities.

  5. Chemical immobilization of North American mule deer

    Science.gov (United States)

    Lange, Robert E.; Nielsen, Leon; Haigh, Jerry C.; Fowler, Murray E.

    1983-01-01

    The choice of agents for chemical immobilization of mule deer (Odocoileus hemionus) is a problem with a simple and effective solution, in my opinion. I recommend combinations of etorphine hydrochloride (M199©) and xylazine hydrochloride (Rompun©) administered intravenously and reversed intravenously. I have used this combination on hundreds of mule deer and have supervised its use on hundreds more. It is a forgiving combination in terms of safety to the deer. I have never seen a mortality in mule deer that I could blame on this combination of drugs, which, in my experience, has performed well under a wide variety of environmental, physiological and organizational conditions.

  6. Immobilization of Uranium Silicides in Sintered Glass

    International Nuclear Information System (INIS)

    Mateos, P.; Russo, D.O.; Heredia, A.D.; Sanfilippo, M.

    2003-01-01

    High activity nuclear spent fuels vitrification by fusion is a well known technology which has industrial scale in France, England, Japan, EEUU. Borosilicates glasses are used in this process.Sintered glasses are an alternative to the immobilization task in which there is also a wide experience around the world.The available technics are: cold pressing and sintering , hot-pressing and hot isostatic pressing.This work compares Borosilicates and Iron silicates sintered glasses behaviour when different ammounts of nuclear simulated waste is added

  7. Methods and devices for protein assays

    Science.gov (United States)

    Chhabra, Swapnil [San Jose, CA; Cintron, Jose M [Indianapolis, IN; Shediac, Renee [Oakland, CA

    2009-11-03

    Methods and devices for protein assays based on Edman degradation in microfluidic channels are disclosed herein. As disclosed, the cleaved amino acid residues may be immobilized in an array format and identified by detectable labels, such as antibodies, which specifically bind given amino acid residues. Alternatively, the antibodies are immobilized in an array format and the cleaved amino acids are labeled identified by being bound by the antibodies in the array.

  8. Two weeks of one-leg immobilization decreases skeletal muscle respiratory capacity equally in young and elderly men

    DEFF Research Database (Denmark)

    Gram, Martin; Vigelsø Hansen, Andreas; Yokota, Takashi

    2014-01-01

    Physical inactivity affects human skeletal muscle mitochondrial oxidative capacity but the influence of aging combined with physical inactivity is not known. This study investigates the effect of two weeks of immobilization followed by six weeks of supervised cycle training on muscle oxidative...... capacity in 17 young (23±1years) and 15 elderly (68±1years) healthy men. We applied high-resolution respirometry in permeabilized fibers from muscle biopsies at inclusion after immobilization and training. Furthermore, protein content of mitochondrial complexes I-V, mitochondrial heat shock protein 70 (mt......HSP70) and voltage dependent anion channel (VDAC) were measured in skeletal muscle by Western blotting. The elderly men had lower content of complexes I-V and mtHSP70 but similar respiratory capacity and content of VDAC compared to the young. In both groups the respiratory capacity and protein content...

  9. Antiretroviral therapy provided to HIV-infected Malawian women in a randomized trial diminishes the posiitive effect of lipid-based nutrient supplements on breast milk B-vitamins

    Science.gov (United States)

    Background: There is little information on B-vitamin concentrations in human milk or how they are affected by maternal B-vitamin deficiencies, antiretroviral (ARV) therapy or maternal supplementation. Objective: To evaluate effects of ARV therapy and/or lipid-based nutrient supplements (LNS) on B-v...

  10. Effect of supplementation with a lipid-based nutrient supplement on the micronutrient status of children aged 6–18 months living in the rural region of Intibucá, Honduras

    Science.gov (United States)

    Background: Lipid-based nutrient supplements (LNS) have been effective in the treatment of acute malnutrition among children. We evaluated the use of LNS supplementation for improving the micronutrient status of young children. Methods: A 12-month randomised controlled trial was conducted among chil...

  11. Maternal cortisol and stress are associated with birth outcomes, but are not affected by lipid-based micronutrient supplements during pregnancy: an analysis of data from a randomized controlled trial in rural Malawi

    Science.gov (United States)

    Background: Prenatal micronutrient supplements have been found to increase birth weight, but mechanisms for increased growth are poorly understood. Our hypotheses were that 1) women who receive lipid-based micronutrient supplements (LNS) during pregnancy would have lower salivary cortisol concentrat...

  12. Frozen Microemulsions for MAPLE Immobilization of Lipase

    Directory of Open Access Journals (Sweden)

    Valeria Califano

    2017-12-01

    Full Text Available Candida rugosa lipase (CRL was deposited by matrix assisted pulsed laser evaporation (MAPLE in order to immobilize the enzyme with a preserved native conformation, which ensures its catalytic functionality. For this purpose, the composition of the MAPLE target was optimized by adding the oil phase pentane to a water solution of the amino acid 3-(3,4-dihydroxyphenyl-2-methyl-l-alanine (m-DOPA, giving a target formed by a frozen water-lipase-pentane microemulsion. Fourier transform infrared (FTIR spectroscopy and atomic force microscopy (AFM were used to investigate the structure of MAPLE deposited lipase films. FTIR deconvolution of amide I band indicated a reduction of unfolding and aggregation, i.e., a better preserved lipase secondary structure in the sample deposited from the frozen microemulsion target. AFM images highlighted the absence of big aggregates on the surface of the sample. The functionality of the immobilized enzyme to promote transesterification was determined by thin layer chromatography, resulting in a modified specificity.

  13. Plutonium immobilization in glass and ceramics

    International Nuclear Information System (INIS)

    Knecht, D.A.; Murphy, W.M.

    1996-01-01

    The Materials Research Society Nineteenth Annual Symposium on the Scientific Basis for Nuclear Waste Management was held in Boston on November 27 to December 1, 1995. Over 150 papers were presented at the Symposium dealing with all aspects of nuclear waste management and disposal. Fourteen oral sessions and on poster session included a Plenary session on surplus plutonium dispositioning and waste forms. The proceedings, to be published in April, 1996, will provide a highly respected, referred compilation of the state of scientific development in the field of nuclear waste management. This paper provides a brief overview of the selected Symposium papers that are applicable to plutonium immobilization and plutonium waste form performance. Waste forms that were described at the Symposium cover most of the candidate Pu immobilization options under consideration, including borosilicate glass with a melting temperature of 1150 degrees C, a higher temperature (1450 degrees C) lanthanide glass, single phase ceramics, multi-phase ceramics, and multi-phase crystal-glass composites (glass-ceramics or slags). These Symposium papers selected for this overview provide the current status of the technology in these areas and give references to the relevant literature

  14. Glass forms for immobilization of Hanford wastes

    International Nuclear Information System (INIS)

    Schulz, W.W.; Dressen, A.L.; Hobbick, C.W.; Babad, H.

    1975-03-01

    Approximately 140 million liters of solid salt cake (mainly NaNO 3 ), produced by evaporation of aged alkaline high-level liquid wastes, will be stored in underground tanks when the present Hanford Waste Management Program is completed in the early 1980's. At this time also, large volumes of various other solid radioactive wastes (sludges, excavated Pu-contaminated soil, and doubly encapsulated 137 CsCl and 90 SrF 2 ) will be stored on the Hanford Reservation. All these solid wastes can be converted to immobile silicate and aluminosilicate glasses of low water leachability by melting them at 1100 0 to 1400 0 C with appropriate amounts of basalt (or sand) and other glass-formers such as B 2 O 3 or CaO. Reviewed in this paper are formulations and other melt conditions used successfully in batch tests to make glasses from actual and synthetic wastes; leachability and other properties of these glasses show them to be satisfactory vehicles for immobilization of the Hanford wastes. (U.S.)

  15. Electrode-immobilized compounds through γ radiation

    International Nuclear Information System (INIS)

    De Castro, E.S.

    1983-01-01

    Chemically Modified Electrodes (CMEs) are used as substrates in heterogeneous catalysis and as sensors. This work demonstrates a new strategy for immobilizing polyelectrolytes and electroactive agents on electrode surfaces. The success of this method lies in cross-linking water soluble polymer chains through the ionizing radiation of γ emissions from a 60 Co source. Cross-linking can create a continuous network out of the polymer macromolecules which then makes the network insoluble on the electrode surface. Bonds between the network and the substrate are also possible. Redox species mixed with the polymer network and irradiated become part of the insoluble network, and are permanently attached. The use of γ radiation to make electrochemical sensors is demonstrated. The immobilized network poly[diallyl dimethyl ammonium chloride] (DDAC) is placed in a solution of potassium ferricyanide and ionicly exchanges the anion into the network. An electroactive network is created from irradiating a mixture of DDAC and 2,6-dichlorophenolindophenol (DCIP). Using the amount of electroactive DCIP remaining in the film as the optimization parameter, variables such as polymer:DCIP ratio, film thickness, and dosage employed are shown to be relevant

  16. Plutonium immobilization in glass and ceramics

    Energy Technology Data Exchange (ETDEWEB)

    Knecht, D.A. [Lockheed Martin Idaho Technologies, Idaho Falls (United States); Murphy, W.M. [Southwest Research Institute, San Antonio, TX (United States)

    1996-05-01

    The Materials Research Society Nineteenth Annual Symposium on the Scientific Basis for Nuclear Waste Management was held in Boston on November 27 to December 1, 1995. Over 150 papers were presented at the Symposium dealing with all aspects of nuclear waste management and disposal. Fourteen oral sessions and on poster session included a Plenary session on surplus plutonium dispositioning and waste forms. The proceedings, to be published in April, 1996, will provide a highly respected, referred compilation of the state of scientific development in the field of nuclear waste management. This paper provides a brief overview of the selected Symposium papers that are applicable to plutonium immobilization and plutonium waste form performance. Waste forms that were described at the Symposium cover most of the candidate Pu immobilization options under consideration, including borosilicate glass with a melting temperature of 1150 {degrees}C, a higher temperature (1450 {degrees}C) lanthanide glass, single phase ceramics, multi-phase ceramics, and multi-phase crystal-glass composites (glass-ceramics or slags). These Symposium papers selected for this overview provide the current status of the technology in these areas and give references to the relevant literature.

  17. Plutonium Immobilization Project - Robotic canister loading

    International Nuclear Information System (INIS)

    Hamilton, R.L.

    2000-01-01

    The Plutonium Immobilization Program (PIP) is a joint venture between the Savannah River Site (SRS), Lawrence Livermore National Laboratory (LLNL), Argonne National Laboratory (ANL), and Pacific Northwest National Laboratory (PNNL). When operational in 2008, the PIP will fulfill the nation's nonproliferation commitment by placing surplus weapons-grade plutonium in a permanently stable ceramic form and making it unattractive for reuse. Since there are significant radiation and security concerns, the program team is developing novel and unique technology to remotely perform plutonium immobilization tasks. The remote task covered in this paper employs a jointed arm robot to load seven 3.5 inch diameter, 135-pound cylinders (magazines) through the 4 inch diameter neck of a stainless steel canister. Working through the narrow canister neck, the robot secures the magazines into a specially designed rack pre-installed in the canister. To provide the deterrent effect, the canisters are filled with a mixture of high-level waste and glass at the Defense Waste Processing Facility (DWPF)

  18. In Situ Immobilization of Selenium in Sediment

    Energy Technology Data Exchange (ETDEWEB)

    Moore, Robert C. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Stewart, Thomas Austin [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2014-09-01

    This project focused on the use of a sorbent, carbonated apatite, to immobilize selenium in the environment. It is know that apatite will sorb selenium and based on the mechanism of sorption it is theorized that carbonated apatite will be more effective that pure apatite. Immobilization of selenium in the environment is through the use of a sorbent in a permeable reactive barrier (PRB). A PRB can be constructed by trenching and backfill with the sorbent or in the case of apatite as the sorbent formed in situ using the apatite forming solution of Moore (2003, 2004). There is very little data on selenium sorption by carbonated apatite in the literature. Therefore, in this work, the basic sorptive properties of carbonated apatite were investigated. Carbonated apatite was synthesized by a precipitation method and characterized. Batch selenium kinetic and equilibrium experiments were performed. The results indicate the carbonated apatite contained 9.4% carbonate and uptake of selenium as selenite was rapid; 5 hours for complete uptake of selenium vs. more than 100 hours for pure hydroxyapatite reported in the literature. Additionally, the carbonated apatite exhibited significantly higher distribution coefficients in equilibrium experiments than pure apatite under similar experimental conditions. The next phase of this work will be to seek additional funds to continue the research with the goal of eventually demonstrating the technology in a field application.

  19. Immobilization of IFR salt wastes in mortar

    International Nuclear Information System (INIS)

    Fischer, D.F.; Johnson, T.R.

    1988-01-01

    Portland cement-base mortars are being considered for immobilizing chloride salt wastes produced by the fuel cycles of Integral Fast Reactors (IFR). The IFR is a sodium-cooled fast reactor with metal alloy fuels. It has a close-coupled fuel cycle in which fission products are separated from the actinides in an electrochemical cell operating at 500/degree/C. This cell has a liquid cadmium anode in which the fuels are dissolved and a liquid salt electrolyte. The salt will be a mixture of either lithium, potassium, and sodium chlorides or lithium, calcium, barium, and sodium chlorides. One method being considered for immobilizing the treated nontransuranic salt waste is to disperse the salt in a portland cement-base mortar that will be sealed in corrosion-resistant containers. For this application, the grout must be sufficiently fluid that it can be pumped into canister-molds where it will solidify into a strong, leach-resistant material. The set times must be longer than a few hours to allow sufficient time for processing, and the mortar must reach a reasonable compressive strength (/approximately/7 MPa) within three days to permit handling. Because fission product heating will be high, about 0.6 W/kg for a mortar containing 10% waste salt, the effects of elevated temperatures during curing and storage on mortar properties must be considered

  20. Nanoparticle-based capillary electroseparation of proteins in polymer capillaries under physiological conditions

    DEFF Research Database (Denmark)

    Nilsson, C.; Harwigsson, I.; Becker, K.

    2010-01-01

    Totally porous lipid-based liquid crystalline nanoparticles were used as pseudostationary phase for capillary electroseparation with LIF detection of proteins at physiological conditions using unmodified cyclic olefin copolymer capillaries (Topas (R), 6.7 cm effective length). In the absence of n...... at protein friendly conditions. The developed capillary-based method facilitates future electrochromatography of proteins on polymer-based microchips under physiological conditions and enables the initial optimization of separation conditions in parallel to the chip development....