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.

Optical Biosensor with Multienzyme System Immobilized onto Hybrid Membrane for Pesticides Determination

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Lyubov Yotova

2011-12-01

Full Text Available A construction of optical biosensor based on simultaneous immobilization of acetylcholinesterase and choline oxidase enzymes for the detection of pesticides residues is described. Different kinds of novel SiO2 hybrid membranes were synthesized to be suitable for optical biosensors using sol-gel techniques. The bioactive component of the sensor consists of a multi-enzyme system including acetylcholinesterase and choline oxidase covalently immobilized on new hybrid membranes. The sensor exhibited a linear response to acetylcholine in a concentration range of 2.5 - 30 mM. Inhibition plots obtained from testing carbamate (carbofuran pesticides exhibited concentration dependent behaviour and showed linear profiles in concentration ranges between 5x10-8 - 5x10-7 M for carbofuran. The factors affecting the constructed optical biosensors were investigated.

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.

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

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.

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

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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.

An Antibody-Immobilized Silica Inverse Opal Nanostructure for Label-Free Optical Biosensors

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Wang Sik Lee

2018-01-01

Full Text Available Three-dimensional SiO2-based inverse opal (SiO2-IO nanostructures were prepared for use as biosensors. SiO2-IO was fabricated by vertical deposition and calcination processes. Antibodies were immobilized on the surface of SiO2-IO using 3-aminopropyl trimethoxysilane (APTMS, a succinimidyl-[(N-maleimidopropionamido-tetraethyleneglycol] ester (NHS-PEG4-maleimide cross-linker, and protein G. The highly accessible surface and porous structure of SiO2-IO were beneficial for capturing influenza viruses on the antibody-immobilized surfaces. Moreover, as the binding leads to the redshift of the reflectance peak, the influenza virus could be detected by simply monitoring the change in the reflectance spectrum without labeling. SiO2-IO showed high sensitivity in the range of 103–105 plaque forming unit (PFU and high specificity to the influenza A (H1N1 virus. Due to its structural and optical properties, SiO2-IO is a promising material for the detection of the influenza virus. Our study provides a generalized sensing platform for biohazards as various sensing strategies can be employed through the surface functionalization of three-dimensional nanostructures.

An Antibody-Immobilized Silica Inverse Opal Nanostructure for Label-Free Optical Biosensors.

Science.gov (United States)

Lee, Wang Sik; Kang, Taejoon; Kim, Shin-Hyun; Jeong, Jinyoung

2018-01-20

Three-dimensional SiO₂-based inverse opal (SiO₂-IO) nanostructures were prepared for use as biosensors. SiO₂-IO was fabricated by vertical deposition and calcination processes. Antibodies were immobilized on the surface of SiO₂-IO using 3-aminopropyl trimethoxysilane (APTMS), a succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester (NHS-PEG₄-maleimide) cross-linker, and protein G. The highly accessible surface and porous structure of SiO₂-IO were beneficial for capturing influenza viruses on the antibody-immobilized surfaces. Moreover, as the binding leads to the redshift of the reflectance peak, the influenza virus could be detected by simply monitoring the change in the reflectance spectrum without labeling. SiO₂-IO showed high sensitivity in the range of 10³-10⁵ plaque forming unit (PFU) and high specificity to the influenza A (H1N1) virus. Due to its structural and optical properties, SiO₂-IO is a promising material for the detection of the influenza virus. Our study provides a generalized sensing platform for biohazards as various sensing strategies can be employed through the surface functionalization of three-dimensional nanostructures.

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.

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)

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

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

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.

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.

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

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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

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

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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.

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.

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.

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

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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

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.

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.

Fiber optic-based biosensor

Science.gov (United States)

Ligler, Frances S.

1991-01-01

The NRL fiber optic biosensor is a device which measures the formation of a fluorescent complex at the surface of an optical fiber. Antibodies and DNA binding proteins provide the mechanism for recognizing an analyze and immobilizing a fluorescent complex on the fiber surface. The fiber optic biosensor is fast, sensitive, and permits analysis of hazardous materials remote from the instrumentation. The fiber optic biosensor is described in terms of the device configuration, chemistry for protein immobilization, and assay development. A lab version is being used for assay development and performance characterization while a portable device is under development. Antibodies coated on the fiber are stable for up to two years of storage prior to use. The fiber optic biosensor was used to measure concentration of toxins in the parts per billion (ng/ml) range in under a minute. Immunoassays for small molecules and whole bacteria are under development. Assays using DNA probes as the detection element can also be used with the fiber optic sensor, which is currently being developed to detect biological warfare agents, explosives, pathogens, and toxic materials which pollute the environment.

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...

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

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.

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)

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.

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.

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

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...

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.

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.

Light assisted drying (LAD) for protein stabilization: optical characterization of samples

Science.gov (United States)

Young, Madison A.; McKinnon, Madison E.; Elliott, Gloria D.; Trammell, Susan R.

2018-02-01

Light-Assisted Drying (LAD) is a novel biopreservation technique which allows proteins to be immobilized in a dry, amorphous solid at room temperature. Indicator proteins are used in a variety of diagnostic assays ranging from highthroughput 96-well plates to new microfluidic devices. A challenge in the development of protein-based assays is preserving the structure of the protein during production and storage of the assay, as the structure of the protein is responsible for its functional activity. Freeze-drying or freezing are currently the standard for the preservation of proteins, but these methods are expensive and can be challenging in some environments due to a lack of available infrastructure. An inexpensive, simple processing method that enables supra-zero temperature storage of proteins used in assays is needed. Light-assisted drying offers a relatively inexpensive method for drying samples. Proteins suspended in a trehalose solution are dehydrated using near-infrared laser light. The laser radiation speeds drying and as water is removed the sugar forms a protective matrix. The goal of this study is optically characterize samples processed with LAD. We use polarized light imaging (PLI) to look at crystallization kinetics of samples and determine optimal humidity. PLI shows a 62.5% chance of crystallization during LAD processing and negligible crystallization during low RH storage.

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.

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.

Development of a Novel Optical Biosensor for Detection of Organophoshorus Pesticides Based on Methyl Parathion Hydrolase Immobilized by Metal-Chelate Affinity

Science.gov (United States)

Lan, Wensheng; Chen, Guoping; Cui, Feng; Tan, Feng; Liu, Ran; Yushupujiang, Maolidan

2012-01-01

We have developed a novel optical biosensor device using recombinant methyl parathion hydrolase (MPH) enzyme immobilized on agarose by metal-chelate affinity to detect organophosphorus (OP) compounds with a nitrophenyl group. The biosensor principle is based on the optical measurement of the product of OP catalysis by MPH (p-nitrophenol). Briefly, MPH containing six sequential histidines (6× His tag) at its N-terminal was bound to nitrilotriacetic acid (NTA) agarose with Ni ions, resulting in the flexible immobilization of the bio-reaction platform. The optical biosensing system consisted of two light-emitting diodes (LEDs) and one photodiode. The LED that emitted light at the wavelength of the maximum absorption for p-nitrophenol served as the signal light, while the other LED that showed no absorbance served as the reference light. The optical sensing system detected absorbance that was linearly correlated to methyl parathion (MP) concentration and the detection limit was estimated to be 4 μM. Sensor hysteresis was investigated and the results showed that at lower concentration range of MP the difference got from the opposite process curves was very small. With its easy immobilization of enzymes and simple design in structure, the system has the potential for development into a practical portable detector for field applications. PMID:23012501

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

Development of a Novel Optical Biosensor for Detection of Organophoshorus Pesticides Based on Methyl Parathion Hydrolase Immobilized by Metal-Chelate Affinity

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Wensheng Lan

2012-06-01

Full Text Available We have developed a novel optical biosensor device using recombinant methyl parathion hydrolase (MPH enzyme immobilized on agarose by metal-chelate affinity to detect organophosphorus (OP compounds with a nitrophenyl group. The biosensor principle is based on the optical measurement of the product of OP catalysis by MPH (p-nitrophenol. Briefly, MPH containing six sequential histidines (6× His tag at its N-terminal was bound to nitrilotriacetic acid (NTA agarose with Ni ions, resulting in the flexible immobilization of the bio-reaction platform. The optical biosensing system consisted of two light-emitting diodes (LEDs and one photodiode. The LED that emitted light at the wavelength of the maximum absorption for p-nitrophenol served as the signal light, while the other LED that showed no absorbance served as the reference light. The optical sensing system detected absorbance that was linearly correlated to methyl parathion (MP concentration and the detection limit was estimated to be 4 μM. Sensor hysteresis was investigated and the results showed that at lower concentration range of MP the difference got from the opposite process curves was very small. With its easy immobilization of enzymes and simple design in structure, the system has the potential for development into a practical portable detector for field applications.

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

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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.

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

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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.

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.

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

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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

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

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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

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)

Covalent immobilization of rabbit-antiaflatoxin-antibodies onto the poly-acrylamideacrylonitrile as well as hybrid material UREASIL and developing an optical immunosensor

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Slavova M.

2017-03-01

Full Text Available The aim of this work is to describe a covalent immobilization of antibodies onto the poly- acrylamide-acrylonitrile or hybrid material UREASIL and creation of optical immunosensor for determination of aflatoxin Bl. For this purpose, mouse-anti-aflatoxin B1 antibodies with oxidized carbohydrate moieties were covalently immobilized on the membranes of polyacrylamide- polyacrylonitrile copolymer, as well as the hybrid material UREASIL. To determine the affinity> binding of the immobilized antibody with afatoxin Bl was used "sandwich" method. Associated with the immobilized antibody sought ingredients interact with a surplus of secondary’ signal antibodies. The described method has been developed as a model system, which can easily be applied for the determination of aflatoxins in samples of different origin. To the best of our knowledge, this is the first study to show that in the establishment of biosensor was used hybrid material UREASIL.

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...

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

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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

Engineering cholesterol-based fibers for antibody immobilization and cell capture

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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

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.

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.

(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.

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.

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.

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

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.

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

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.

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.

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.

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.

Design of an optically stable pH sensor based on immobilization of Giemsa on triacetylcellulose membrane.

Science.gov (United States)

Khodadoust, Saeid; Kouri, Narges Cham; Talebiyanpoor, Mohammad Sharif; Deris, Jamile; Pebdani, Arezou Amiri

2015-12-01

In this work a simple, inexpensive, and sensitive optical sensor based on triacetylcellulose membrane as solid support was developed by using immobilization of Giemsa indicator for pH measurement. In this method, the influence variables on the membrane performance including pH concentration of indicator, response time, ionic strength, and reversibility were investigated. At optimum values of all variables the response of optical pH sensor is linear in the pH range of 3.0-12.0. This optical sensor was produced through simultaneous binding of the Giemsa on the activated triacetylcellulose membrane which responded to the pH changes in a broader linear range within less than 2.0 min and suitable reproducibility (RSDsensor was stable after 6 months of storage in the water/ethanol (50:50, v/v) solution without any measurable divergence in response properties (less than 5% RSD). Copyright © 2015 Elsevier B.V. All rights reserved.

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.

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

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.

Spontaneous stacking of purple membranes during immobilization with physical cross-linked poly(vinyl alcohol) hydrogel with retaining native-like functionality of bacteriorhodopsin

Science.gov (United States)

Yokoyama, Yasunori; Tanaka, Hikaru; Yano, Shunsuke; Takahashi, Hiroshi; Kikukawa, Takashi; Sonoyama, Masashi; Takenaka, Koshi

2017-05-01

We previously discovered the correlation between light-induced chromophore color change of a photo-receptor membrane protein bacteriorhodopsin (bR) and its two-dimensional crystalline state in the membrane. To apply this phenomenon to a novel optical memory device, it is necessary that bR molecules are immobilized as maintaining their structure and functional properties. In this work, a poly(vinyl alcohol) (PVA) hydrogel with physical cross-linkages (hydrogen bonds between PVA chains) that resulted from repeated freezing-and-thawing (FT) cycles was used as an immobilization medium. To investigate the effects of physically cross-linked PVA gelation on the structure and function of bR in purple membranes (PMs), spectroscopic techniques were employed against PM/PVA immobilized samples prepared with different FT cycle numbers. Visible circular dichroism spectroscopy strongly suggested PM stacking during gelation. X-ray diffraction data also indicated the PM stacking as well as its native-like crystalline lattice even after gelation. Time-resolved absorption spectroscopy showed that bR photocycle behaviors in PM/PVA immobilized samples were almost identical to that in suspension. These results suggested that a physically cross-linked PVA hydrogel is appropriate for immobilizing membrane proteins in terms of maintaining their structure and functionality.

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

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.

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.

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.

Customization of Protein Single Nanowires for Optical Biosensing.

Science.gov (United States)

Sun, Yun-Lu; Sun, Si-Ming; Wang, Pan; Dong, Wen-Fei; Zhang, Lei; Xu, Bin-Bin; Chen, Qi-Dai; Tong, Li-Min; Sun, Hong-Bo

2015-06-24

An all-protein single-nanowire optical biosensor is constructed by a facile and general femtosecond laser direct writing approach with nanoscale structural customization. As-formed protein single nanowires show excellent optical properties (fine waveguiding performance and bio-applicable transmission windows), and are utilized as evanescent optical nanobiosensors for label-free biotin detection. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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.

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.

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.

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.

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.

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)

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...

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...

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.

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...

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.

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

A preliminary investigation on the interaction between sol-gel immobilized glucose oxidase and freely diffusing glucose by means of two-photon microscopy

Science.gov (United States)

Delfino, I.; Portaccio, M.; De Rosa, M.; Lepore, M.

2013-02-01

To study immobilized protein interactions with dissolved substrates is a very important topic both from a fundamental and technological standpoint. In the present report we illustrate the preliminary results obtained on sol-gel immobilized glucose oxidase (GOD) using a standard de-scanned two-photon microscope based on a modified confocal scanhead with internal detectors and a Ti:sapphire laser as a source. Data acquisition conditions were preliminary defined using functionalized beads of different dimensions. Various sol-gel supports were then investigated by monitoring endogeneous fluorescence due to the flavoadenine (FAD) molecules, present in GOD. Linear absorption and fluorescence spectroscopy along with Fourier Transform Infrared microscopy were employed for a full-optical characterization of the samples. The results show that GOD immobilization processes can be successfully monitored in some cases and also the interaction with glucose could be studied by this approach. This assessment holds potentials to better understand the characteristic of immobilized enzymes biocatalysis and to develop new biosensing schemes.

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.

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

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

Immobilization of biomolecules onto surfaces according to ultraviolet light diffraction patterns

DEFF Research Database (Denmark)

Petersen, Steffen B.; Gennaro, Ane Kold Di; Neves Petersen, Teresa

2010-01-01

We developed a method for immobilization of biomolecules onto thiol functionalized surfaces according to UV diffraction patterns. UV light-assisted molecular immobilization proceeds through the formation of free, reactive thiol groups that can bind covalently to thiol reactive surfaces. We demons......, with a fine structured interference pattern superimposed. (C) 2010 Optical Society of America...

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.

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.

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.

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.

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

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.

Label-free detection of C-reactive protein using reflectometric interference spectroscopy-based sensing system

Energy Technology Data Exchange (ETDEWEB)

Choi, Hyung Woo; Sakata, Yasuhiko [Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501 (Japan); Kurihara, Yoshikazu [Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501 (Japan); KONICA MINOLTA OPTO, Inc., 1 Sakura-machi, Hino-shi, Tokyo 191-8511 (Japan); Ooya, Tooru [Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501 (Japan); Takeuchi, Toshifumi, E-mail: takeuchi@gold.kobe-u.ac.jp [Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501 (Japan)

2012-05-30

Highlights: Black-Right-Pointing-Pointer A new RIfS-based label-free biosensing system for C-reactive protein was developed. Black-Right-Pointing-Pointer Silicon-based inexpensive chips and the simple optical setup were employed. Black-Right-Pointing-Pointer Owing to the TMS treatment and the use of protein A, the sensitivity was enhanced. Black-Right-Pointing-Pointer It can be applied to other target as a substitute of SPR-based expensive sensors. - Abstract: Reflectometric interference spectroscopy (RIfS) is a label-free, time-resolved technique, and suitable for detecting antibody-antigen interaction. This work describes a continuous flow biosensor for C-reactive protein (CRP), involving an effective immobilization method of a monoclonal antibody against CRP (anti-CRP) to achieve highly sensitive RIfS-based detection of CRP. The silicon nitride-coated silicon chip (SiN chip) for the RIfS sensing was first treated with trimethylsilylchloride (TMS), followed by UV-light irradiation to in situ generation of homogeneous silanols on the surface. Following amination by 3-aminopropyltriethoxysilane, carboxymethyldextran (CMD) was grafted, and subsequently, protein A was immobilized to create the oriented anti-CRP surface. The immobilization process of protein A and anti-CRP was monitored with the RIfS system by consecutive injections of an amine coupling reagent, protein A and anti-CRP, respectively, to confirm the progress of each step in real time. The sensitivity was enhanced when all of the processes were adopted, suggesting that the oriented immobilization of anti-CRP via protein A that was coupled with the grafted CMD on the aminated surface of TMS-treated SiN chip. The feasibility of the present sensing system was demonstrated on the detection of CRP, where the silicon-based inexpensive chips and the simple optical setup were employed. It can be applied to other target molecules in various fields of life science as a substitute of surface plasmon resonance

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.

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.

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

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.

SU-F-J-18: Feasibility of Open Mask Immobilization with Optical Imaging Guidance (OIG) for H&N Radiotherapy

International Nuclear Information System (INIS)

Zhao, B; Maquilan, G; Anders, M; Jiang, S; Schwartz, D

2016-01-01

Purpose: Full face and neck thermoplastic masks provide standard-of-care immobilization for patients receiving H&N IMRT. However, these masks are uncomfortable and increase skin dose. The purpose of this pilot study was to investigate the feasibility and setup accuracy of open face and neck mask immobilization with OIG. Methods: Ten patients were consented and enrolled to this IRB-approved protocol. Patients were immobilized with open masks securing only forehead and chin. Standard IMRT to 60–70 Gy in 30 fractions were delivered in all cases. Patient simulation information, including isocenter location and CT skin contours, were imported to a commercial OIG system. On the first day of treatment, patients were initially set up to surface markings and then OIG referenced to face and neck skin regions of interest (ROI) localized on simulation CT images, followed by in-room CBCT. CBCTs were acquired at least weekly while planar OBI was acquired on the days without CBCT. Following 6D robotic couch correction with kV imaging, a new optical real-time surface image was acquired to track intrafraction motion and to serve as a reference surface for setup at the next treatment fraction. Therapists manually recorded total treatment time as well as couch shifts based on kV imaging. Intrafractional ROI motion tracking was automatically recorded. Results: Setup accuracy of OIG was compared with CBCT results. The setup error based on OIG was represented as a 6D shift (vertical/longitudinal/lateral/rotation/pitch/roll). Mean error values were −0.70±3.04mm, −0.69±2.77mm, 0.33±2.67 mm, −0.14±0.94 o, −0.15±1.10o and 0.12±0.82o, respectively for the cohort. Average treatment time was 24.1±9.2 minutes, comparable to standard immobilization. The amplitude of intrafractional ROI motion was 0.69±0.36 mm, driven primarily by respiratory neck motion. Conclusion: OGI can potentially provide accurate setup and treatment tracking for open face and neck immobilization. Study

SU-F-J-18: Feasibility of Open Mask Immobilization with Optical Imaging Guidance (OIG) for H&N Radiotherapy

Energy Technology Data Exchange (ETDEWEB)

Zhao, B; Maquilan, G; Anders, M; Jiang, S; Schwartz, D [UT Southwestern Medical Center, Dallas, TX (United States)

2016-06-15

Purpose: Full face and neck thermoplastic masks provide standard-of-care immobilization for patients receiving H&N IMRT. However, these masks are uncomfortable and increase skin dose. The purpose of this pilot study was to investigate the feasibility and setup accuracy of open face and neck mask immobilization with OIG. Methods: Ten patients were consented and enrolled to this IRB-approved protocol. Patients were immobilized with open masks securing only forehead and chin. Standard IMRT to 60–70 Gy in 30 fractions were delivered in all cases. Patient simulation information, including isocenter location and CT skin contours, were imported to a commercial OIG system. On the first day of treatment, patients were initially set up to surface markings and then OIG referenced to face and neck skin regions of interest (ROI) localized on simulation CT images, followed by in-room CBCT. CBCTs were acquired at least weekly while planar OBI was acquired on the days without CBCT. Following 6D robotic couch correction with kV imaging, a new optical real-time surface image was acquired to track intrafraction motion and to serve as a reference surface for setup at the next treatment fraction. Therapists manually recorded total treatment time as well as couch shifts based on kV imaging. Intrafractional ROI motion tracking was automatically recorded. Results: Setup accuracy of OIG was compared with CBCT results. The setup error based on OIG was represented as a 6D shift (vertical/longitudinal/lateral/rotation/pitch/roll). Mean error values were −0.70±3.04mm, −0.69±2.77mm, 0.33±2.67 mm, −0.14±0.94 o, −0.15±1.10o and 0.12±0.82o, respectively for the cohort. Average treatment time was 24.1±9.2 minutes, comparable to standard immobilization. The amplitude of intrafractional ROI motion was 0.69±0.36 mm, driven primarily by respiratory neck motion. Conclusion: OGI can potentially provide accurate setup and treatment tracking for open face and neck immobilization. Study

High Throughput, Label-free Screening Small Molecule Compound Libraries for Protein-Ligands using Combination of Small Molecule Microarrays and a Special Ellipsometry-based Optical Scanner.

Science.gov (United States)

Landry, James P; Fei, Yiyan; Zhu, X D

2011-12-01

Small-molecule compounds remain the major source of therapeutic and preventative drugs. Developing new drugs against a protein target often requires screening large collections of compounds with diverse structures for ligands or ligand fragments that exhibit sufficiently affinity and desirable inhibition effect on the target before further optimization and development. Since the number of small molecule compounds is large, high-throughput screening (HTS) methods are needed. Small-molecule microarrays (SMM) on a solid support in combination with a suitable binding assay form a viable HTS platform. We demonstrate that by combining an oblique-incidence reflectivity difference optical scanner with SMM we can screen 10,000 small-molecule compounds on a single glass slide for protein ligands without fluorescence labeling. Furthermore using such a label-free assay platform we can simultaneously acquire binding curves of a solution-phase protein to over 10,000 immobilized compounds, thus enabling full characterization of protein-ligand interactions over a wide range of affinity constants.

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…

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...

Immobilization of cholesterol oxidase on magnetic fluorescent core-shell-structured nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Huang, Jun, E-mail: hjun@whut.edu.cn; Liu, Huichao; Zhang, Peipei; Zhang, Pengfei; Li, Mengshi; Ding, Liyun

2015-12-01

The magnetic fluorescent core-shell structured nanoparticles, Fe{sub 3}O{sub 4}@SiO{sub 2}(F)@meso–SiO{sub 2} nanoparticles, were prepared. Cholesterol oxidase (COD) was immobilized on their surface to form Fe{sub 3}O{sub 4}@SiO{sub 2}(F)@meso–SiO{sub 2}@COD nanoparticles. Optimal immobilization was achieved with 2.5% (v/v) APTES, 2.0% (v/v) GA, 10 mg COD (in 15 mg carrier) and solution pH of 7.0. Fe{sub 3}O{sub 4}@SiO{sub 2}(F)@meso–SiO{sub 2}@COD nanoparticles showed maximal catalytic activity at pH 7.0 and 50 °C. The thermal, storage and operational stabilities of COD were improved greatly after its immobilization. After the incubation at 50 °C for 5 h, the nanoparticles and free COD retained 80% and 46% of its initial activity, respectively. After kept at 4 °C for 30 days, the nanoparticles and free COD maintained 86% and 65% of initial activity, respectively. The nanoparticles retained 71% of its initial activity after 7 consecutive operations. Since Fe{sub 3}O{sub 4}@SiO{sub 2}(F)@meso–SiO{sub 2}@COD nanoparticles contained tris(2,2-bipyridyl)dichloro-ruthenium(II) hexahydrate (Ru(bpy){sub 3}Cl{sub 2}) and were optical sensitive to oxygen in solution, it might be used as the sensing material and has the application potential in multi parameter fiber optic biosensor based on enzyme catalysis and oxygen consumption. - Highlights: • COD was immobilized on magnetic fluorescent core-shell structured nanoparticles. • The nanoparticles were optical sensitive to oxygen in water solution. • The nanoparticles have remarkable improved stability compared with free COD. • The nanoparticles can probably be used in multi parameter fiber optic Biosensor.

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.

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.

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.

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.

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.

A microstructured Polymer Optical Fiber Biosensor

DEFF Research Database (Denmark)

Emiliyanov, Grigoriy Andreev; Jensen, Jesper Bo; Hoiby, Poul E.

2006-01-01

We demonstrate selective detection of fluorophore labeled antibodies from minute samples probed by a sensor layer of the complementary biomolecules immobilized inside the air holes of microstructured Polymer Optical Fibers.......We demonstrate selective detection of fluorophore labeled antibodies from minute samples probed by a sensor layer of the complementary biomolecules immobilized inside the air holes of microstructured Polymer Optical Fibers....

Fluorescence-Based Multiplex Protein Detection Using Optically Encoded Microbeads

Directory of Open Access Journals (Sweden)

Dae Hong Jeong

2012-03-01

Full Text Available Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based multiplex detection techniques. Among the various techniques for high-throughput protein screening, optically-encoded beads combined with fluorescence-based target monitoring have great advantages over the planar array-based multiplexing assays. This review discusses recent developments of analytical methods of screening protein molecules on microbead-based platforms. These include various strategies such as barcoded microbeads, molecular beacon-based techniques, and surface-enhanced Raman scattering-based techniques. Their applications for label-free protein detection are also addressed. Especially, the optically-encoded beads such as multilayer fluorescence beads and SERS-encoded beads are successful for generating a large number of coding.

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...

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

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

[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.

A DNA-Mediated Homogeneous Binding Assay for Proteins and Small Molecules

DEFF Research Database (Denmark)

Zhang, Zhao; Hejesen, Christian; Kjelstrup, Michael Brøndum

2014-01-01

. The shift occurs upon binding of a protein, for example, an antibody to its target. We demonstrate nanomolar detection of small molecules such as biotin, digoxigenin, vitamin D, and folate, in buffer and in plasma. The method is flexible, and we also show nanomolar detection of the respective antibodies......Optical detection of molecular targets typically requires immobilization, separation, or chemical or enzymatic processing. An important exception is aptamers that allow optical detection in solution based on conformational changes. This method, however, requires the laborious selection of aptamers...

Optical fiber chemiluminescence sensor for iron (II) ion based on immobilized luminol

International Nuclear Information System (INIS)

Alipao, Arthur A.; Sevilla, Fortunato III.

1999-01-01

A chemiluminescence (CL) sensor for iron (II) was developed based on the catalytic action of the analyte on the CL reaction between luminol and oxygen. The reagents were immobilized on a cellulose membrane and set on a reaction cell which was coupled by means of an optical fiber to a spectrofluorometer. The concentration of iron(II) was quantified by measuring the intensity of the light generated from the CL reaction. The response of the sensor system was rapid and highly reproducible. Good sensitivity was displayed by the sensor system over the five orders of magnitude of iron(II) ion concentration. The calibration curve consisted of two portions: (1) a linear range at lower concentrations (7.5 x 10 -7 M to 1.0 x 10 -4 M) exhibiting a positive slope arising from a catalytic action, and (2) a linear range at higher concentrations (5.0 x 10 -2 M to 2.5 x 10 -4 M) wherein the slope is negative due to an inhibitory action of iron(II) on the CL reaction. The sensor system was highly selective for iron(II) ions. (Author)

Preparative resolution of D,L-threonine catalyzed by immobilized phosphatase.

Science.gov (United States)

Scollar, M P; Sigal, G; Klibanov, A M

1985-03-01

Hydrolysis of L- and D-O-phosphothreonines catalyzed by four different phosphatases, alkaline phosphatases from calf intestine and E. coli and acid phosphatases from wheat germ and potato, has been kinetically studied. Alkaline phosphatases were found to have comparable reactivities towards the optical isomers. On the other hand, both acid phosphatases displayed a marked stereoselectivity, hydrolyzing the L-ester much faster than its D counterpart. Wheat germ acid phosphatase was the most stereoselective enzyme: V(L)/V(D) = 24 and K(m,L)/K(m,D) = 0.17. This enzyme was immobilized (in k-carrageenan gel, followed by crosslinking with glutaraldehyde) and used for the preparative resolution of D,L-threonine: the latter was first chemically O-phosphorylated and then asymmetrically hydrolyzed by the immobilized phosphatase. As a result, gram quantities of L-threonine of high optical purity and O-phospho-D-threonine were prepared. Immobilized wheat germ phosphatase has been tested for the resolution of other racemic alcohols: serine, 2-amino-1-butanol, 1-amino-2-propanol, 2-octanol, and menthol. In all those cases, the enzyme was either not sufficiently stereoselective or too slow for preparative resolutions.

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

Hybrid system of semiconductor and photosynthetic protein

International Nuclear Information System (INIS)

Kim, Younghye; Shin, Seon Ae; Lee, Jaehun; Yang, Ki Dong; Nam, Ki Tae

2014-01-01

Photosynthetic protein has the potential to be a new attractive material for solar energy absorption and conversion. The development of semiconductor/photosynthetic protein hybrids is an example of recent progress toward efficient, clean and nanostructured photoelectric systems. In the review, two biohybrid systems interacting through different communicating methods are addressed: (1) a photosynthetic protein immobilized semiconductor electrode operating via electron transfer and (2) a hybrid of semiconductor quantum dots and photosynthetic protein operating via energy transfer. The proper selection of materials and functional and structural modification of the components and optimal conjugation between them are the main issues discussed in the review. In conclusion, we propose the direction of future biohybrid systems for solar energy conversion systems, optical biosensors and photoelectric devices. (topical reviews)

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.

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.

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.

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.

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.

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.

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

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

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...

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.

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

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.

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.

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.

In vivo phosphorylation of axonal proteins in goldfish optic nerve during regeneration

Energy Technology Data Exchange (ETDEWEB)

Larrivee, D.C.; Grafstein, B.

1987-01-01

In vivo phosphorylation of axonal proteins was investigated in normal and regenerating optic nerves of goldfish by two-dimensional gel electrophoresis. By 6-24 h after intraocular injection of H/sub 3/(32)PO/sub 4/, approximately 20 optic nerve proteins ranging in size from 19 to 180 kilodaltons and in pI from 4.4 to 6.8 were seen to have incorporated radiolabel. Five of these proteins showed a robust increase in incorporation of phosphate during regeneration. Among the latter was an acidic (pI 4.5) 45-kilodalton protein, which has previously been shown to be conveyed by fast axonal transport and to increase dramatically in its rate of synthesis during regeneration of goldfish optic axons.

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.

SU-E-T-603: Analysis of Optical Tracked Head Inter-Fraction Movements Within Masks to Access Intracranial Immobilization Techniques in Proton Therapy

International Nuclear Information System (INIS)

Hsi, W; Zeidan, O

2014-01-01

Purpose: We present a quantitative methodology utilizing an optical tracking system for monitoring head inter-fraction movements within brain masks to assess the effectiveness of two intracranial immobilization techniques. Methods and Materials: A 3-point-tracking method was developed to measure the mask location for a treatment field at each fraction. Measured displacement of mask location to its location at first fraction is equivalent to the head movement within the mask. Head movements for each of treatment fields were measured over about 10 fractions at each patient for seven patients; five treated in supine and two treated in prone. The Q-fix Base-of-Skull head frame was used in supine while the CIVCO uni-frame baseplate was used in prone. Displacements of recoded couch position of each field post imaging at each fraction were extracted for those seven patients. Standard deviation (S.D.) of head movements and couch displacements was scored for statistical analysis. Results: The accuracy of 3PtTrack method was within 1.0 mm by phantom measurements. Patterns of head movement and couch displacement were similar for patients treated in either supine or prone. In superior-inferior direction, mean value of scored standard deviations over seven patients were 1.6 mm and 3.4 mm for the head movement and the couch displacement, respectively. The result indicated that the head movement combined with a loose fixation between the mask-to-head frame results large couch displacements for each patient, and also large variation between patients. However, the head movement is the main cause for the couch displacement with similar magnitude of around 1.0 mm in anterior-posterior and lateral directions. Conclusions: Optical-tracking methodology independently quantifying head movements could improve immobilization devices by correctly acting on causes for head motions within mask. A confidence in the quality of intracranial immobilization techniques could be more efficient by

Graphene oxide as a protein matrix: influence on protein biophysical properties.

Science.gov (United States)

Hernández-Cancel, Griselle; Suazo-Dávila, Dámaris; Ojeda-Cruzado, Axel J; García-Torres, Desiree; Cabrera, Carlos R; Griebenow, Kai

2015-10-19

This study provides fundamental information on the influence of graphene oxide (GO) nanosheets and glycans on protein catalytic activity, dynamics, and thermal stability. We provide evidence of protein stabilization by glycans and how this strategy could be implemented when GO nanosheets is used as protein immobilization matrix. A series of bioconjugates was constructed using two different strategies: adsorbing or covalently attaching native and glycosylated bilirubin oxidase (BOD) to GO. Bioconjugate formation was followed by FT-IR, zeta-potential, and X-ray photoelectron spectroscopy measurements. Enzyme kinetic parameters (k(m) and k(cat)) revealed that the substrate binding affinity was not affected by glycosylation and immobilization on GO, but the rate of enzyme catalysis was reduced. Structural analysis by circular dichroism showed that glycosylation did not affect the tertiary or the secondary structure of BOD. However, GO produced slight changes in the secondary structure. To shed light into the biophysical consequence of protein glycosylation and protein immobilization on GO nanosheets, we studied structural protein dynamical changes by FT-IR H/D exchange and thermal inactivation. It was found that glycosylation caused a reduction in structural dynamics that resulted in an increase in thermostability and a decrease in the catalytic activity for both, glycoconjugate and immobilized enzyme. These results establish the usefulness of chemical glycosylation to modulate protein structural dynamics and stability to develop a more stable GO-protein matrix.

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.

Rapid and label-free detection of protein a by aptamer-tethered porous silicon nanostructures.

Science.gov (United States)

Urmann, Katharina; Reich, Peggy; Walter, Johanna-Gabriela; Beckmann, Dieter; Segal, Ester; Scheper, Thomas

2017-09-10

Protein A, which is secreted by and displayed on the cell membrane of Staphylococcus aureus is an important biomarker for S. aureus. Thus, its rapid and specific detection may facilitate the pathogen identification and initiation of proper treatment. Herein, we present a simple, label-free and rapid optical biosensor enabling specific detection of protein A. Protein A-binding aptamer serves as the capture probe and is immobilized onto a nanostructured porous silicon thin film, which serves as the optical transducer element. We demonstrate high sensitivity of the biosensor with a linear detection range between 8 and 23μM. The apparent dissociation constant was determined as 13.98μM and the LoD is 3.17μM. Harnessing the affinity between protein A and antibodies, a sandwich assay format was developed to amplify the optical signal associated with protein A capture by the aptamer. Using this approach, we increase the sensitivity of the biosensor, resulting in a three times lower LoD. Copyright © 2017 Elsevier B.V. All rights reserved.

Well-oriented ZZ–PS-tag with high Fc-binding onto polystyrene surface for controlled immobilization of capture antibodies

International Nuclear Information System (INIS)

Tang, Jin-Bao; Sun, Xi-Feng; Yang, Hong-Ming; Zhang, Bao-Gang; Li, Zhi-Jian; Lin, Zhi-Juan; Gao, Zhi-Qin

2013-01-01

Graphical abstract: -- Highlights: •A versatile platform for immobilizing functionally intact IgG is proposed. •The mechanism relies on properly oriented ZZ–PS-tag onto a hydrophilic PS surface. •The oriented ZZ–PS-tag presents ∼fivefold higher IgG-binding activity. •The platform shows tenfold higher sensitivity and a wider linear range in ELISA. -- Abstract: The site specificity and bioactivity retention of antibodies immobilized on a solid substrate are crucial requirements for solid phase immunoassays. A fusion protein between an immunoglobulin G (IgG)-binding protein (ZZ protein) and a polystyrene-binding peptide (PS-tag) was constructed, and then used to develop a simple method for the oriented immobilization of the ZZ protein onto a PS support by the specific attachment of the PS-tag onto a hydrophilic PS. The orientation of intact IgG was achieved via the interaction of the ZZ protein and the constant fragment (Fc), thereby displayed the Fab fragment for binding antigen. The interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP was analyzed. Results showed that the oriented ZZ–PS-tag yielded an IgG-binding activity that is fivefold higher than that produced by the passive immobilization of the ZZ protein. The advantage of the proposed immunoassay strategy was demonstrated through an enzyme-linked immunosorbent assay, in which monoclonal mouse anti-goat IgG and HRP-conjugated rabbit F(ab′) 2 anti-goat IgG were used to detect goat IgG. The ZZ–PS-tag presented a tenfold higher sensitivity and a wider linear range than did the passively immobilized ZZ protein. The proposed approach may be an attractive strategy for a broad range of applications involving the oriented immobilization of intact IgGs onto PS supports, in which only one type of phi-PS (ZZ–PS-tag) surface is used

Well-oriented ZZ–PS-tag with high Fc-binding onto polystyrene surface for controlled immobilization of capture antibodies

Energy Technology Data Exchange (ETDEWEB)

Tang, Jin-Bao, E-mail: tangjinbao@yahoo.com.cn [School of Pharmacy and Biology, Weifang Medical University, Weifang 261053 (China); Sun, Xi-Feng [Clinical Laboratory, Weifang People' s Hospital, Weifang 261041 (China); Yang, Hong-Ming [School of Pharmacy and Biology, Weifang Medical University, Weifang 261053 (China); Zhang, Bao-Gang [School of Basic Medicine, Weifang Medical University, Weifang 261053 (China); Li, Zhi-Jian [School of Pharmacy and Biology, Weifang Medical University, Weifang 261053 (China); Lin, Zhi-Juan [School of Basic Medicine, Weifang Medical University, Weifang 261053 (China); Gao, Zhi-Qin, E-mail: zhiqingao@yahoo.cn [School of Pharmacy and Biology, Weifang Medical University, Weifang 261053 (China)

2013-05-07

Graphical abstract: -- Highlights: •A versatile platform for immobilizing functionally intact IgG is proposed. •The mechanism relies on properly oriented ZZ–PS-tag onto a hydrophilic PS surface. •The oriented ZZ–PS-tag presents ∼fivefold higher IgG-binding activity. •The platform shows tenfold higher sensitivity and a wider linear range in ELISA. -- Abstract: The site specificity and bioactivity retention of antibodies immobilized on a solid substrate are crucial requirements for solid phase immunoassays. A fusion protein between an immunoglobulin G (IgG)-binding protein (ZZ protein) and a polystyrene-binding peptide (PS-tag) was constructed, and then used to develop a simple method for the oriented immobilization of the ZZ protein onto a PS support by the specific attachment of the PS-tag onto a hydrophilic PS. The orientation of intact IgG was achieved via the interaction of the ZZ protein and the constant fragment (Fc), thereby displayed the Fab fragment for binding antigen. The interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP was analyzed. Results showed that the oriented ZZ–PS-tag yielded an IgG-binding activity that is fivefold higher than that produced by the passive immobilization of the ZZ protein. The advantage of the proposed immunoassay strategy was demonstrated through an enzyme-linked immunosorbent assay, in which monoclonal mouse anti-goat IgG and HRP-conjugated rabbit F(ab′){sub 2} anti-goat IgG were used to detect goat IgG. The ZZ–PS-tag presented a tenfold higher sensitivity and a wider linear range than did the passively immobilized ZZ protein. The proposed approach may be an attractive strategy for a broad range of applications involving the oriented immobilization of intact IgGs onto PS supports, in which only one type of phi-PS (ZZ–PS-tag) surface is used.

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

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

Highly selective and sensitive optical sensor for determination of Pb2+and Hg2+ ions based on the covalent immobilization of dithizone on agarose membrane

Science.gov (United States)

Zargoosh, Kiomars; Babadi, Fatemeh Farhadian

2015-02-01

A highly sensitive and selective optical membrane for determination of Hg2+ and Pb2+ was prepared by covalent immobilization of dithizone on agarose membrane. In addition to its high stability, reproducibility and relatively long lifetime, the proposed optical sensor revealed good selectivity for target ions over a large number of alkali, alkaline earth, transition, and heavy metal ions. The proposed optical membrane displays linear responses from 1.1 × 10-8 to 2.0 × 10-6 mol L-1 and 1.2 × 10-8 to 2.4 × 10-6 mol L-1 for Hg2+ and Pb2+, respectively. The limits of detection (LOD) were 2.0 × 10-9 mol L-1 and 4.0 × 10-9 mol L-1 for Hg2+ and Pb2, respectively. The prepared optical membrane was successfully applied to the determination of Hg2+ and Pb2+ in industrial wastes, spiked tap water and natural waters without any preconcentration step.

Adsorption properties of BSA and DsRed proteins deposited on thin SiO2 layers: optically non-absorbing versus absorbing proteins

Science.gov (United States)

Scarangella, A.; Soumbo, M.; Villeneuve-Faure, C.; Mlayah, A.; Bonafos, C.; Monje, M.-C.; Roques, C.; Makasheva, K.

2018-03-01

Protein adsorption on solid surfaces is of interest for many industrial and biomedical applications, where it represents the conditioning step for micro-organism adhesion and biofilm formation. To understand the driving forces of such an interaction we focus in this paper on the investigation of the adsorption of bovine serum albumin (BSA) (optically non-absorbing, model protein) and DsRed (optically absorbing, naturally fluorescent protein) on silica surfaces. Specifically, we propose synthesis of thin protein layers by means of dip coating of the dielectric surface in protein solutions with different concentrations (0.01-5.0 g l-1). We employed spectroscopic ellipsometry as the most suitable and non-destructive technique for evaluation of the protein layers’ thickness and optical properties (refractive index and extinction coefficient) after dehydration, using two different optical models, Cauchy for BSA and Lorentz for DsRed. We demonstrate that the thickness, the optical properties and the wettability of the thin protein layers can be finely controlled by proper tuning of the protein concentration in the solution. These results are correlated with the thin layer morphology, investigated by AFM, FTIR and PL analyses. It is shown that the proteins do not undergo denaturation after dehydration on the silica surface. The proteins arrange themselves in a lace-like network for BSA and in a rod-like structure for DsRed to form mono- and multi-layers, due to different mechanisms driving the organization stage.

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.

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

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...

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

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.

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.

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.

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.

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.

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.

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...

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)

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

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.

Laser-based optical activity detection of amino acids and proteins

Energy Technology Data Exchange (ETDEWEB)

Reitsma, B.H.

1987-08-01

The optical activity detector (OAD) for HPLC is a selective detector for optically active substances including amino acids and proteins. Four free amino acids were resolved using cation-exchange chromatography followed by detection with refractive index detector (RI) for proline and threonine and the OAD to an ultraviolet absorbance detector (uv) for tyrosine and phenylalanine. Amino acid detection by refractive index is not sensitive and uv absorbance detects only three amino acids. Derivatization of amino acids to make them detectable by uv absorbance enhances the applicability of OA/uv for the determination of enantiomeric ratios. The separation of 16 dansyl-L-amino acids by RP-HPLC with detection by OA/uv is illustrated. Calculation of the specific rotation of 22 dansyl-L-amino acids shows that derivatization enhances the OA detectability of some amino acids but degrades that of others. RP-HPLC of proteins is a rapidly developing technique. Several researchers have reported the detection of multiple peaks when a pure protein is subjected to HPLC under certain conditions. These multiple peaks have been determined to be different conformations of the same protein. Since proteins are optically active, OA is a suitable detector. The RP-HPLC separation of conformers of soybean trypsin inhibitor is illustrated. Detection by OA/uv provides insights from the chromatogram unavailable from uv absorbance detection alone. In addition, identification of impurities is simplified with OA/uv. Specific rotations of the separated protein fractions show no significant change accompanying change in conformation. 163 refs., 13 figs., 9 tabs.

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.

Immobilized xylenol orange as a sensitive element for fiber-optic sensors for thorium(4) and lead(2). Immobilizovannyj ksilenolovyj oranzhevyj kak chuvstvitel'nyj ehlement dlya volokonno-opticheskikh sensorov na torij(4) i svinets(2)

Energy Technology Data Exchange (ETDEWEB)

Trutneva, L M; Shvoeva, O P; Savvin, S B

1989-10-01

A new type of carrier for immobilization of reagent-fibrous materials of polyacrylonitrile fiber filled with fine-dispersed ion exchanger during formation- was suggested when creating sensitive element of optical sensor for lead (2) and thorium(4). The highest sensitivity and contrast are achieved in color reactions with xylenol orange, immobilized on weak-base anion-exchanger. Detection limits are decreased down to 2x10{sup -8}M, the range of determined concentrations -5x10{sup -8}-1x10{sup -6} M, reproducibility Sr 0.05-0.10, the response forms during 1-10 minutes.

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.

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.

1-butanethiol vapor sensing based on gold nanoparticle immobilized on glass slide by digital color analysis

Science.gov (United States)

Shokoufi, Nader; Adeleh, Sara

2017-12-01

We demonstrate that gold nanoparticles (GNPs) immobilized on silanized glass act as an optical sensor that is able to quantify 1-butanethiol vapor. GNPs optical properties in the visible region are dominated by the surface plasmon resonance (SPR). The high affinity between 1-butanethiol and GNPs through Au-s bond leads to change in plasmon feature of GNPs that immobilized on silanized glass and causes absorption decrease at 542 nm in SPR spectrum of GNPs. It can be used as an optical sensor for quantitative detection. In this research, the glass slide surface activated by aminopropyltriethoxysilane (APTES). Spherical GNPs immobilized on silanized glass by silanization agent. The sensor is based on the spectrophotometry and digital color analysis (DCA) through RGB. We monitored R value and linear range 50-700 µM (R 2  =  0.97) with 2.05% relative standard deviation and 26.5 µM value was achieved, for the limit of detection. This method represents advantages of metal gold nanoparticles and solid substrate stability in one package, being inexpensive and low time consuming is another advantage of our method that can be conducted in petrochemical, pharmaceutical industries, and for detection of rotten food in food industries.

F-18 Labeled Diabody-Luciferase Fusion Proteins for Optical-ImmunoPET

Energy Technology Data Exchange (ETDEWEB)

Wu, Anna M. [Univ. of California, Los Angeles, CA (United States)

2013-01-18

The goal of the proposed work is to develop novel dual-labeled molecular imaging probes for multimodality imaging. Based on small, engineered antibodies called diabodies, these probes will be radioactively tagged with Fluorine-18 for PET imaging, and fused to luciferases for optical (bioluminescence) detection. Performance will be evaluated and validated using a prototype integrated optical-PET imaging system, OPET. Multimodality probes for optical-PET imaging will be based on diabodies that are dually labeled with 18F for PET detection and fused to luciferases for optical imaging. 1) Two sets of fusion proteins will be built, targeting the cell surface markers CEA or HER2. Coelenterazine-based luciferases and variant forms will be evaluated in combination with native substrate and analogs, in order to obtain two distinct probes recognizing different targets with different spectral signatures. 2) Diabody-luciferase fusion proteins will be labeled with 18F using amine reactive [18F]-SFB produced using a novel microwave-assisted, one-pot method. 3) Sitespecific, chemoselective radiolabeling methods will be devised, to reduce the chance that radiolabeling will inactivate either the target-binding properties or the bioluminescence properties of the diabody-luciferase fusion proteins. 4) Combined optical and PET imaging of these dual modality probes will be evaluated and validated in vitro and in vivo using a prototype integrated optical-PET imaging system, OPET. Each imaging modality has its strengths and weaknesses. Development and use of dual modality probes allows optical imaging to benefit from the localization and quantitation offered by the PET mode, and enhances the PET imaging by enabling simultaneous detection of more than one probe.

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.

Optical Sensors for Biomolecules Using Nanoporous Sol-Gel Materials

Science.gov (United States)

Fang, Jonathan; Zhou, Jing C.; Lan, Esther H.; Dunn, Bruce; Gillman, Patricia L.; Smith, Scott M.

2004-01-01

An important consideration for space missions to Mars is the ability to detect biosignatures. Solid-state sensing elements for optical detection of biological entities are possible using sol-gel based biologically active materials. We have used these materials as optical sensing elements in a variety of bioassays, including immunoassays and enzyme assays. By immobilizing an appropriate biomolecule in the sol-gel sensing element, we have successfully detected analytes such as amino acids and hormones. In the case of the amino acid glutamate, the enzyme glutamate dehydrogenase was the immobilized molecule, whereas in the case of the hormone cortisol, an anti-cortisol antibody was immobilized in the sensing element. In this previous work with immobilized enzymes and antibodies, excellent sensitivity and specificity were demonstrated in a variety of formats including bulk materials, thin films and fibers. We believe that the sol-gel approach is an attractive platform for bioastronautics sensing applications because of the ability to detect a wide range of entities such as amino acids, fatty acids, hopanes, porphyrins, etc. The sol-gel approach produces an optically transparent 3D silica matrix that forms around the biomolecule of interest, thus stabilizing its structure and functionality while allowing for optical detection. This encapsulation process protects the biomolecule and leads to a more "rugged" sensor. The nanoporous structure of the sol-gel matrix allows diffusion of small target molecules but keeps larger, biomolecules immobilized in the pores. We are currently developing these biologically active sol-gel materials into small portable devices for on-orbit cortisol detection

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.

Studies on immobilized porphyrinderivatives for the development of cadmium-sensitive layers to be used in an optical-chemical sensor

International Nuclear Information System (INIS)

Czolk, R.

1991-02-01

The aim of this work was to develop an optical-chemical sensor for the reversible detection of cadmium by immobilizing a reagent dye onto or into a polymeric matrix. Using the water-soluble 5,10,15,20-meso-tetra(p-sulphonatophenyl)porphyrin (TPPS), a spectrophotometric method was developed for the detection of cadmium(II)-ions in aqueous, alkaline solutions (pH = 9,0; duration of complexation: 20 minutes; detection limit: c min = 6x10 -8 mol/l). As a result of the different absorption spectra of various metal-porphyrin species selectivity is increased when statistical evaluation methods are used. VA-Epoxy - a cross-linked poly(vinylacetate) - and poly(vinylchloride) were used as polymeric matrices. The absorbance of six different sensors produced with the VA-Epoxy-powder shows a standard deviation of 6.2%. TPPS is immobilized with a spacermolecule (1,3-diaminopropane). The response time is about the same as in an aqueous solution. Complexation is reversible. The long-term stability is about 7 days (decrease in absorbance by 7.1%). The optimum pH-value for the complexation is shifted into the alkaline region. The detection limit of the sensor is c min = 13 μmol/l. The immobilized dye also shows the characteristic absorption spectra of various metal-porphyrin-complexes. The thickness of produced PVC-layers on a glassplatelet was less than 10 μm (standard deviation: 17.4%; n=26). Sensors with the hydrophobic 5,10,15,20-meso-tetraphenylporphyrin (TPP) have a long-term stability of 12 days and a response time of more than 8 hours. Sensors with the hydrophilic TPPS attain a response time of about 20 minutes and show a constant absorbance of the cadmium-complex already above pH = 6.5. The detection limit is c min = 1.9x10 -5 mol/l. The long-term stability is limited by the leaching of the dye. (orig./BBR) [de

Ultra-Thin Optically Transparent Carbon Electrodes Produced from Layers of Adsorbed Proteins

Science.gov (United States)

Alharthi, Sarah A.; Benavidez, Tomas E.; Garcia, Carlos D.

2013-01-01

This work describes a simple, versatile, and inexpensive procedure to prepare optically transparent carbon electrodes, using proteins as precursors. Upon adsorption, the protein-coated substrates were pyrolyzed under reductive conditions (5% H2) to form ultra-thin, conductive electrodes. Because proteins spontaneously adsorb to interfaces forming uniform layers, the proposed method does not require a precise control of the preparation conditions, specialized instrumentation, or expensive precursors. The resulting electrodes were characterized by a combination of electrochemical, optical, and spectroscopic means. As a proof-of-concept, the optically-transparent electrodes were also used as substrate for the development of an electrochemical glucose biosensor. The proposed films represent a convenient alternative to more sophisticated, and less available, carbon-based nanomaterials. Furthermore, these films could be formed on a variety of substrates, without classical limitations of size or shape. PMID:23421732

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

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.

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.

Covalent Immobilization of Peroxidase onto Hybrid Membranes for the Construction of Optical Biosensor

Directory of Open Access Journals (Sweden)

Lyubov Yotova

2015-06-01

Full Text Available The aim of this study is to covalently immobilize horse radish peroxidase (HRP onto new hybrid membranes synthesized by the sol-gel method based on silica precursors, dendrimers and cellulose derivatives. This new system will be used for designing biosensor. For investigation of the properties of membranes, HRP was used as a modeling enzyme. Kinetic parameters, pH and temperature optimum were determined, and the structure of the membranes surface was examined. Results showed higher relative and residual activity of HRP immobilized onto membranes with cellulose acetate butyrate with high molecular weight CAB/H. This novel biosensor could offer a simple, cheap and rapid tool with enhanced sensing performance as well as having potentials to find application in medicine, pharmacy, food and process control and environmental monitoring.

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.

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.

Chondroitin sulfate immobilization at the surface of electrospun nanofiber meshes for cartilage tissue regeneration approaches

International Nuclear Information System (INIS)

Piai, Juliana Francis; Alves da Silva, Marta; Martins, Albino; Torres, Ana Bela; Faria, Susana

2017-01-01

Highlights: • Chemical immobilization of chondroitin sulfate at the surface of nanofiber meshes. • CS-immobilized NFMs showed lower roughness and higher hydrophilicity. • CS-immobilized NFMs offer a highly effective substrate for hACs phenotypic stability. - Abstract: Aiming at improving the biocompatibility of biomaterial scaffolds, surface modification presents a way to preserve their mechanical properties and to improve the surface bioactivity. In this work, chondroitin sulfate (CS) was immobilized at the surface of electrospun poly(caprolactone) nanofiber meshes (PCL NFMs), previously functionalized by UV/O_3 exposure and aminolysis. Contact angle, SEM, optical profilometry, FTIR, X-ray photoelectron spectroscopy techniques confirmed the success of CS-immobilization in PCL NFMs. Furthermore, CS-immobilized PCL NFMs showed lower roughness and higher hydrophilicity than the samples without CS. Human articular chondrocytes (hACs) were cultured on electrospun PCL NFMs with or without CS immobilization. It was observed that hACs proliferated through the entire time course of the experiment in both types of nanofibrous scaffolds, as well as for the production of glycosaminoglycans. Quantitative-PCR results demonstrated over-expression of cartilage-related genes such as Aggrecan, Collagen type II, COMP and Sox9 on both types of nanofibrous scaffolds. Morphological observations from SEM and LSCM revealed that hACs maintained their characteristic round shape and cellular agglomeration exclusively on PCL NFMs with CS immobilization. In conclusion, CS immobilization at the surface of PCL NFMs was achieved successfully and provides a valid platform enabling further surface functionalization methods in scaffolds to be developed for cartilage tissue engineering.

Chondroitin sulfate immobilization at the surface of electrospun nanofiber meshes for cartilage tissue regeneration approaches

Energy Technology Data Exchange (ETDEWEB)

Piai, Juliana Francis [3B’s Research Group − Biomaterials, Biodegradables and Biomimetics, Department of Polymer Engineering, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4805-017 Barco, Guimarães (Portugal); ICVS/3B’s − PT Government Associate Laboratory, Braga/Guimarães (Portugal); Grupo de Materiais Poliméricos e Compósitos, GMPC – Departamento de Química- Universidade Estadual de Maringá, Av. Colombo 5790, 87020-900, Maringá, Paraná (Brazil); Alves da Silva, Marta; Martins, Albino; Torres, Ana Bela [3B’s Research Group − Biomaterials, Biodegradables and Biomimetics, Department of Polymer Engineering, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4805-017 Barco, Guimarães (Portugal); ICVS/3B’s − PT Government Associate Laboratory, Braga/Guimarães (Portugal); Faria, Susana [Research Center Officinal Mathematical, Department of Mathematics for Science and Technology, University of Minho, Campus de Azurém, 4800-058 Guimarães (Portugal); and others

2017-05-01

Highlights: • Chemical immobilization of chondroitin sulfate at the surface of nanofiber meshes. • CS-immobilized NFMs showed lower roughness and higher hydrophilicity. • CS-immobilized NFMs offer a highly effective substrate for hACs phenotypic stability. - Abstract: Aiming at improving the biocompatibility of biomaterial scaffolds, surface modification presents a way to preserve their mechanical properties and to improve the surface bioactivity. In this work, chondroitin sulfate (CS) was immobilized at the surface of electrospun poly(caprolactone) nanofiber meshes (PCL NFMs), previously functionalized by UV/O{sub 3} exposure and aminolysis. Contact angle, SEM, optical profilometry, FTIR, X-ray photoelectron spectroscopy techniques confirmed the success of CS-immobilization in PCL NFMs. Furthermore, CS-immobilized PCL NFMs showed lower roughness and higher hydrophilicity than the samples without CS. Human articular chondrocytes (hACs) were cultured on electrospun PCL NFMs with or without CS immobilization. It was observed that hACs proliferated through the entire time course of the experiment in both types of nanofibrous scaffolds, as well as for the production of glycosaminoglycans. Quantitative-PCR results demonstrated over-expression of cartilage-related genes such as Aggrecan, Collagen type II, COMP and Sox9 on both types of nanofibrous scaffolds. Morphological observations from SEM and LSCM revealed that hACs maintained their characteristic round shape and cellular agglomeration exclusively on PCL NFMs with CS immobilization. In conclusion, CS immobilization at the surface of PCL NFMs was achieved successfully and provides a valid platform enabling further surface functionalization methods in scaffolds to be developed for cartilage tissue engineering.

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.

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.

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.

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.

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.

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.).

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.

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

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.

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

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...

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.

Homogeneous protein analysis by magnetic core-shell nanorod probes

KAUST Repository

Schrittwieser, Stefan

2016-03-29

Studying protein interactions is of vital importance both to fundamental biology research and to medical applications. Here, we report on the experimental proof of a universally applicable label-free homogeneous platform for rapid protein analysis. It is based on optically detecting changes in the rotational dynamics of magnetically agitated core-shell nanorods upon their specific interaction with proteins. By adjusting the excitation frequency, we are able to optimize the measurement signal for each analyte protein size. In addition, due to the locking of the optical signal to the magnetic excitation frequency, background signals are suppressed, thus allowing exclusive studies of processes at the nanoprobe surface only. We study target proteins (soluble domain of the human epidermal growth factor receptor 2 - sHER2) specifically binding to antibodies (trastuzumab) immobilized on the surface of our nanoprobes and demonstrate direct deduction of their respective sizes. Additionally, we examine the dependence of our measurement signal on the concentration of the analyte protein, and deduce a minimally detectable sHER2 concentration of 440 pM. For our homogeneous measurement platform, good dispersion stability of the applied nanoprobes under physiological conditions is of vital importance. To that end, we support our measurement data by theoretical modeling of the total particle-particle interaction energies. The successful implementation of our platform offers scope for applications in biomarker-based diagnostics as well as for answering basic biology questions.

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 .

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}.

Protein array staining methods for undefined protein content, manufacturing quality control, and performance validation.

Science.gov (United States)

Schabacker, Daniel S; Stefanovska, Ivana; Gavin, Igor; Pedrak, Casandra; Chandler, Darrell P

2006-12-01

Methods to assess the quality and performance of protein microarrays fabricated from undefined protein content are required to elucidate slide-to-slide variability and interpolate resulting signal intensity values after an interaction assay. We therefore developed several simple total- and posttranslational modification-specific, on-chip staining methods to quantitatively assess the quality of gel element protein arrays manufactured with whole-cell lysate in vitro protein fractions derived from two-dimensional liquid-phase fractionation (PF2D) technology. A linear dynamic range of at least 3 logs was observed for protein stains and immobilized protein content, with a lower limit of detection at 8 pg of protein per gel element with Deep Purple protein stain and a field-portable microarray imager. Data demonstrate the successful isolation, separation, transfer, and immobilization of putative transmembrane proteins from Yersinia pestis KIM D27 with the combined PF2D and gel element array method. Internal bovine serum albumin standard curves provided a method to assess on-chip PF2D transfer and quantify total protein immobilized per gel element. The basic PF2D array fabrication and quality assurance/quality control methods described here therefore provide a standard operating procedure and basis for developing whole-proteome arrays for interrogating host-pathogen interactions, independent of sequenced genomes, affinity tags, or a priori knowledge of target cell composition.

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.

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.

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

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.

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 ...

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.

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...

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.

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...

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.

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.

Development of a novel pH sensor based upon Janus Green B immobilized on triacetyl cellulose membrane: Experimental design and optimization.

Science.gov (United States)

Chamkouri, Narges; Niazi, Ali; Zare-Shahabadi, Vali

2016-03-05

A novel pH optical sensor was prepared by immobilizing an azo dye called Janus Green B on the triacetylcellulose membrane. Condition of the dye solution used in the immobilization step, including concentration of the dye, pH, and duration were considered and optimized using the Box-Behnken design. The proposed sensor showed good behavior and precision (RSDpH range of 2.0-10.0. Advantages of this optical sensor include on-line applicability, no leakage, long-term stability (more than 6 months), fast response time (less than 1 min), high selectivity and sensitivity as well as good reversibility and reproducibility. Copyright © 2015. Published by Elsevier B.V.

Development of a novel pH sensor based upon Janus Green B immobilized on triacetyl cellulose membrane: Experimental design and optimization

Science.gov (United States)

Chamkouri, Narges; Niazi, Ali; Zare-Shahabadi, Vali

2016-03-01

A novel pH optical sensor was prepared by immobilizing an azo dye called Janus Green B on the triacetylcellulose membrane. Condition of the dye solution used in the immobilization step, including concentration of the dye, pH, and duration were considered and optimized using the Box-Behnken design. The proposed sensor showed good behavior and precision (RSD < 5%) in the pH range of 2.0-10.0. Advantages of this optical sensor include on-line applicability, no leakage, long-term stability (more than 6 months), fast response time (less than 1 min), high selectivity and sensitivity as well as good reversibility and reproducibility.

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)

Chondroitin sulfate immobilization at the surface of electrospun nanofiber meshes for cartilage tissue regeneration approaches

Science.gov (United States)

Piai, Juliana Francis; da Silva, Marta Alves; Martins, Albino; Torres, Ana Bela; Faria, Susana; Reis, Rui L.; Muniz, Edvani Curti; Neves, Nuno M.

2017-05-01

Aiming at improving the biocompatibility of biomaterial scaffolds, surface modification presents a way to preserve their mechanical properties and to improve the surface bioactivity. In this work, chondroitin sulfate (CS) was immobilized at the surface of electrospun poly(caprolactone) nanofiber meshes (PCL NFMs), previously functionalized by UV/O3 exposure and aminolysis. Contact angle, SEM, optical profilometry, FTIR, X-ray photoelectron spectroscopy techniques confirmed the success of CS-immobilization in PCL NFMs. Furthermore, CS-immobilized PCL NFMs showed lower roughness and higher hydrophilicity than the samples without CS. Human articular chondrocytes (hACs) were cultured on electrospun PCL NFMs with or without CS immobilization. It was observed that hACs proliferated through the entire time course of the experiment in both types of nanofibrous scaffolds, as well as for the production of glycosaminoglycans. Quantitative-PCR results demonstrated over-expression of cartilage-related genes such as Aggrecan, Collagen type II, COMP and Sox9 on both types of nanofibrous scaffolds. Morphological observations from SEM and LSCM revealed that hACs maintained their characteristic round shape and cellular agglomeration exclusively on PCL NFMs with CS immobilization. In conclusion, CS immobilization at the surface of PCL NFMs was achieved successfully and provides a valid platform enabling further surface functionalization methods in scaffolds to be developed for cartilage tissue engineering.

Highly sensitive optical sensor that detects Hg"2"+ and Cu"2"+ by immobilizing dicarboxylate 1,5-diphenyl-3-thiocarbazone on surface functionalized PVA microspheres

International Nuclear Information System (INIS)

Bai, Xue; Gu, Haixin; Hua, Zulin; Dai, Zhangyan; Yang, Bei; Li, Yulong

2015-01-01

Highlights: • PVA microspheres were chosen as carrier and DDT groups were chosen as chromophores. • The DDT–PVA microspheres could detect Hg"2"+ and Cu"2"+ simultaneously within 120 s. • The DDT–PVA microspheres had excellent detection for Hg"2"+ and Cu"2"+ ions. • The DDT–PVA microspheres had preeminent selectivity and reusability. - Abstract: A novel optical sensor to detect Hg"2"+ and Cu"2"+ is prepared by immobilizing a synthesized dicarboxylate 1,5-diphenyl-3-thiocarbazone (DDT) group on functionalized polyvinyl alcohol (PVA) microspheres. This optical sensor is successfully fabricated by extensive characterization with Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Its colorimetric properties, selectivity, sensitivity, and reversibility are investigated as well. In this sensing system, DDT–PVA selectively recognized multiple heavy metal ions, as indicated by the changes in color from orange to scarlet for Hg"2"+ and from orange to gray for Cu"2"+. In particular, this optical sensor exhibits the most apparent color changes at pH levels of 12 and 2. Hence, Hg"2"+ and Cu"2"+ can be detected in aqueous solution at minimum detection limits of 0.053 and 0.132 μM, respectively, with a UV-vis spectrometer. Furthermore, the sensor can be regenerated by ethylene diamine tetraacetic acid and reused several times. Therefore, the optical sensor can detect Hg"2"+ because of its selectivity, sensitivity, and reversibility.

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.

Investigation of Optical Properties of Biomolecular Materials for Developing a Novel Fiber Optic Biosensor.

Science.gov (United States)

Gao, Harry Hong

1995-01-01

Recently considerable efforts have been devoted to the development of optical biosensors for applications such as environmental monitoring and biomedical technology. The research described in this thesis focuses on the development of a novel fiber optic biosensor system for pesticide detection based on enzyme catalyzed chemiluminescence. To optimize the collection efficiency, the tapering effect of a fiber tip has been studied in different cases of light source distribution utilizing fluorescence technique. Our results indicate that a continuously tapered tip with the largest tapering angle is the most efficient configuration when the light source is in a "thick" layer ({> }1 μm) while a combination tapered tip is the best configuration when the light source is either in a thin layer ({offers the flexibility of controlling the number of enzymes on a fiber surface. Multilayer of alkaline phosphatase have been characterized using various techniques including chemiluminescence, ellipsometry and surface plasma resonance. The results indicated that at least 3 layers of enzyme can be assembled on a fiber surface. With this approach, it is possible to immobilize different kinds of enzyme on a fiber surface for biosensors based on a multi-enzyme system. Based on the studies of tapered tip and immobilization schemes, a novel fiber optic biosensor system for the detection of organophosphorous-based pesticide has been developed. The detection mechanism is pesticide inhibition of alkaline phosphatase catalyzed chemiluminescence. Paraoxon with concentration as low as 167 ppb has been detected. This is the first fiber optic chemiluminescence-based biosensor utilizing tapered tips with enzyme immobilized on the fiber surface and a cooled CCD array detector.

Preparation of polymer brushes grafted graphene oxide by atom transfer radical polymerization as a new support for trypsin immobilization and efficient proteome digestion.

Science.gov (United States)

Guo, Cong; Zhao, Xinyuan; Zhang, Wanjun; Bai, Haihong; Qin, Weijie; Song, Haifeng; Qian, Xiaohong

2017-08-01

Highly efficient protein digestion is one of the key issues in the "bottom-up" strategy-based proteomic studies. Compared with the time-consuming solution-based free protease digestion, immobilized protease digestion offers a promising alternative with obviously improved sample processing throughput. In this study, we proposed a new immobilized protease digestion strategy using two kinds of polymer-grafted graphene oxide (GO) conjugated trypsin. The polymer brush grafted GO was prepared using in situ polymer growth on initiator-functionalized GO using surface-initiated atom transfer radical polymerization (SI-ATRP) and characterized by AFM, TEM, TGA, and XPS. The polymer brush grafted GO supports three-dimensional trypsin immobilization, which not only increases the loading amount but also improves accessibility towards protein substrates. Both of the two types of immobilized trypsin provide 700 times shorter digestion time, while maintaining comparable protein/peptide identification scale compared with that of free trypsin digestion. More interestingly, combined application of the two types of immobilized trypsin with different surface-grafted polymers leads to at least 18.3/31.3% enhancement in protein/peptide identification compared with that obtained by digestion using a single type, indicating the potential of this digestion strategy for deeper proteome coverage using limited mass spectrometer machine hour. We expect these advantages may find valuable application in high throughput clinical proteomic studies, which often involve processing of a large number of samples. Graphical abstract Preparation of polymer brushes grafted and trypsin immobilized graphene oxide and its application in proteome digestion and mass spectrometry identification.

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)

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

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.

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

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.

Silver nanoparticle (AgNPs) doped gum acacia-gelatin-silica nanohybrid: an effective support for diastase immobilization.

Science.gov (United States)

Singh, Vandana; Ahmed, Shakeel

2012-03-01

An effective carrier matrix for diastase alpha amylase immobilization has been fabricated by gum acacia-gelatin dual templated polymerization of tetramethoxysilane. Silver nanoparticle (AgNp) doping to this hybrid could significantly enhance the shelf life of the impregnated enzyme while retaining its full bio-catalytic activity. The doped nanohybrid has been characterized as a thermally stable porous material which also showed multipeak photoluminescence under UV excitation. The immobilized diastase alpha amylase has been used to optimize the conditions for soluble starch hydrolysis in comparison to the free enzyme. The optimum pH for both immobilized and free enzyme hydrolysis was found to be same (pH=5), indicating that the immobilization made no major change in enzyme conformation. The immobilized enzyme showed good performance in wide temperature range (from 303 to 323 K), 323 K being the optimum value. The kinetic parameters for the immobilized, (K(m)=10.30 mg/mL, V(max)=4.36 μmol mL(-1)min(-1)) and free enzyme (K(m)=8.85 mg/mL, V(max)=2.81 μmol mL(-1)min(-1)) indicated that the immobilization improved the overall stability and catalytic property of the enzyme. The immobilized enzyme remained usable for repeated cycles and did not lose its activity even after 30 days storage at 40°C, while identically synthesized and stored silver undoped hybrid lost its ~31% activity in 48 h. Present study revealed the hybrids to be potentially useful for biomedical and optical applications. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

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

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.

High Spatial Resolution Imaging Mass Spectrometry of Human Optic Nerve Lipids and Proteins

Science.gov (United States)

Anderson, David M. G.; Spraggins, Jeffrey M.; Rose, Kristie L.; Schey, Kevin L.

2015-06-01

The human optic nerve carries signals from the retina to the visual cortex of the brain. Each optic nerve is comprised of approximately one million nerve fibers that are organized into bundles of 800-1200 fibers surrounded by connective tissue and supportive glial cells. Damage to the optic nerve contributes to a number of blinding diseases including: glaucoma, neuromyelitis optica, optic neuritis, and neurofibromatosis; however, the molecular mechanisms of optic nerve damage and death are incompletely understood. Herein we present high spatial resolution MALDI imaging mass spectrometry (IMS) analysis of lipids and proteins to define the molecular anatomy of the human optic nerve. The localization of a number of lipids was observed in discrete anatomical regions corresponding to myelinated and unmyelinated nerve regions as well as to supporting connective tissue, glial cells, and blood vessels. A protein fragment from vimentin, a known intermediate filament marker for astrocytes, was observed surrounding nerved fiber bundles in the lamina cribrosa region. S100B was also found in supporting glial cell regions in the prelaminar region, and the hemoglobin alpha subunit was observed in blood vessel areas. The molecular anatomy of the optic nerve defined by MALDI IMS provides a firm foundation to study biochemical changes in blinding human diseases.

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.

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

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.

Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

Science.gov (United States)

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

2014-11-27

In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

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.

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.

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

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.

Specific changes in rapidly transported proteins during regeneration of the goldfish optic nerve

International Nuclear Information System (INIS)

Benowitz, L.I.; Shashoua, V.E.; Yoon, M.G.

1981-01-01

Double labeling methods were used to identify changes in the complement of proteins synthesized in the retinal ganglion cells and transported down the optic nerve during the process of axonal regeneration. Eight to 62 days after goldfish underwent a unilateral optic nerve crush, one eye was labeled with [3H]-, the other with [14C]proline. Control and regenerating optic nerves were dissected out and homogenized together after 5 hr, a time which allowed us to examine selectively membrane-bound components which migrate in the rapid phase of axoplasmic transport. Proteins from the two sides were so-purified and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the 3H and 14C incorporation patterns along the gels revealed a radical shift away from the normal labeling spectrum during regeneration, with selective changes in labeling at particular molecular weights varying over a 3-fold range. Eight days after crushing the optic nerve, the greatest increases in labeling were seen for material with apparent molecular weights of 24,000 to 27,000, 44,000, and 210,000 daltons. These peaks declined thereafter, and on days 29 to 39, the most prominent increases were at 110,000 to 140,000 daltons. These studies indicate a continuously changing pattern in the synthesis and/or degradation of proteins that are rapidly transported down the optic nerve during regeneration and point to molecular species potential significance in the establishment of the visual map upon the brain

Ethanol production by fermentation using immobilized cells of Saccharomyces cerevisiae in cashew apple bagasse.

Science.gov (United States)

Pacheco, Alexandre Monteiro; Gondim, Diego Romão; Gonçalves, Luciana Rocha Barros

2010-05-01

In this work, cashew apple bagasse (CAB) was used for Saccharomyces cerevisiae immobilization. The support was prepared through a treatment with a solution of 3% HCl, and delignification with 2% NaOH was also conducted. Optical micrographs showed that high populations of yeast cells adhered to pre-treated CAB surface. Ten consecutive fermentations of cashew apple juice for ethanol production were carried out using immobilized yeasts. High ethanol productivity was observed from the third fermentation assay until the tenth fermentation. Ethanol concentrations (about 19.82-37.83 g L(-1) in average value) and ethanol productivities (about 3.30-6.31 g L(-1) h(-1)) were high and stable, and residual sugar concentrations were low in almost all fermentations (around 3.00 g L(-1)) with conversions ranging from 44.80% to 96.50%, showing efficiency (85.30-98.52%) and operational stability of the biocatalyst for ethanol fermentation. Results showed that cashew apple bagasse is an efficient support for cell immobilization aiming at ethanol production.

[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.

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.

High quality protein microarray using in situ protein purification

Directory of Open Access Journals (Sweden)

Fleischmann Robert D

2009-08-01

Full Text Available Abstract Background In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC. This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays. Results Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents

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

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.

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.

Design and fabrication of optical chemical sensor for detection of nitroaromatic explosives based on fluorescence quenching of phenol red immobilized poly(vinyl alcohol) membrane.

Science.gov (United States)

Zarei, Ali Reza; Ghazanchayi, Behnam

2016-04-01

The present study developed a new optical chemical sensor for detection of nitroaromatic explosives in liquid phase. The method is based on the fluorescence quenching of phenol red as fluorophore in a poly(vinyl alcohol) (PVA) membrane in the presence of nitroaromatic explosives as quenchers, e.g., 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (2,4-DNT), 4-nitrotoluene (4-NT), 2,4,6-trinitrobenzene (TNB), and nitrobenzene (NB). For chemical immobilization of phenol red in PVA, phenol red reacted with formaldehyde to produce hydroxymethyl groups and then attached to PVA membrane through the hydroxymethyl groups. The optical sensor showed strong quenching of nitroaromatic explosives. A Stern-Volmer graph for each explosive was constructed and showed that the range of concentration from 5.0 × 10(-6) to 2.5 × 10(-4) mol L(-1) was linear for each explosive and sensitivity varied as TNB >TNT>2,4-DNT>NB>4-NT. The response time of the sensor was within 1 min. The proposed sensor showed good reversibility and reproducibility. Copyright © 2015 Elsevier B.V. All rights reserved.

Protein bioelectronics: a review of what we do and do not know

Science.gov (United States)

Bostick, Christopher D.; Mukhopadhyay, Sabyasachi; Pecht, Israel; Sheves, Mordechai; Cahen, David; Lederman, David

2018-02-01

We review the status of protein-based molecular electronics. First, we define and discuss fundamental concepts of electron transfer and transport in and across proteins and proposed mechanisms for these processes. We then describe the immobilization of proteins to solid-state surfaces in both nanoscale and macroscopic approaches, and highlight how different methodologies can alter protein electronic properties. Because immobilizing proteins while retaining biological activity is crucial to the successful development of bioelectronic devices, we discuss this process at length. We briefly discuss computational predictions and their connection to experimental results. We then summarize how the biological activity of immobilized proteins is beneficial for bioelectronic devices, and how conductance measurements can shed light on protein properties. Finally, we consider how the research to date could influence the development of future bioelectronic devices.

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.

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)

Optics for protein microcrystallography using synchrotron and laboratory X-ray sources

International Nuclear Information System (INIS)

Varghese, J.N.; Van Donkelaar, A.; Balaic, D.X.; Barnea, Z.

2000-01-01

Full text: For protein crystallography, a highly-intense focused beam overcomes a serious constraint in current biological research: the inability of many protein molecules to form crystals larger than a few tens of microns in size. High structure-resolution X-ray diffraction analysis of microcrystals is currently only being studied at synchrotron X-ray sources. We shall examine how this is being carried out, and also report the development of a novel tapered glass monocapillary toroidal-mirror optic, which achieves a high-intensity, low-divergence focused beam from a rotating-anode Xray generator. We have used this optic, which demonstrates an ∼28x intensity gain at the beam focus to solve the structure of a plant exoglucanse/inhibtor complex microcrystal to 2.8 Angstroms, with volume equivalent to a 30-micron-edge cube

Recent Advances in Immobilization Strategies for Glycosidases

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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

Challenges in the Development of Functional Assays of Membrane Proteins

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Sophie Demarche

2012-11-01

Full Text Available Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

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

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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.

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

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.

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.

Stability of a fiber optic pH sensor at 100 degree F

International Nuclear Information System (INIS)

Angel, S.M.; Northrup, M.A.

1993-02-01

A simple ratiometric fiber-optic pH sensor was developed and accelerated aging studies were performed in 100 degree F distilled water. A ph-sensitive fluorescent indicator dye, HPTS (hydroxypyrenetrisulfonic acid) was convalently attached, using a procedure that was developed during this work, to a polyacrylamide polymer that was subsequently immobilized at the end of an optical fiber. Different immobilization techniques were compared and it was found that physically attaching the indicator gels to the fibers gave the most reproducible long-term results. These fiber-optic sensors were found to give linear pH responses, between pH 6 and 8, and resolution greater than 0.25 pH unit with useful lifetimes exceeding one year

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.

Production of D-alanine from DL-alanine using immobilized cells of Bacillus subtilis HLZ-68.

Science.gov (United States)

Zhang, Yangyang; Li, Xiangping; Zhang, Caifei; Yu, Xiaodong; Huang, Fei; Huang, Shihai; Li, Lianwei; Liu, Shiyu

2017-09-13

Immobilized cells of Bacillus subtilis HLZ-68 were used to produce D-alanine from DL-alanine by asymmetric degradation. Different compounds such as polyvinyl alcohol and calcium alginate were employed for immobilizing the B. subtilis HLZ-68 cells, and the results showed that cells immobilized using a mixture of these two compounds presented higher L-alanine degradation activity, when compared with free cells. Subsequently, the effects of different concentrations of polyvinyl alcohol and calcium alginate on L-alanine consumption were examined. Maximum L-alanine degradation was exhibited by cells immobilized with 8% (w/v) polyvinyl alcohol and 2% (w/v) calcium alginate. Addition of 400 g of DL-alanine (200 g at the beginning of the reaction and 200 g after 30 h of incubation) into the reaction solution at 30 °C, pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm resulted in complete L-alanine degradation within 60 h, leaving 185 g of D-alanine in the reaction solution. The immobilized cells were applied for more than 15 cycles of degradation and a maximum utilization rate was achieved at the third cycle. D-alanine was easily extracted from the reaction solution using cation-exchange resin, and the chemical and optical purity of the extracted D-alanine was 99.1 and 99.6%, respectively.

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.

Montmorillonite Supported Titanium/Antimony Catalyst:Preparation, Characterization and Immobilization

Institute of Scientific and Technical Information of China (English)

CHEN Guiyong; WANG Xiaoqun; ZHAO Chuan; DU Shanyi

2014-01-01

Montmorillonite supported titanium (Ti-MMT) or antimony catalyst (Sb-MMT) has been a hot area of research on preparing polyethylene terephthalate/montmorillonite (PET/MMT) nanocomposites by in situ polymerization. So removal of Ti or Sb from Ti-MMT or Sb-MMT is not expected during in situ polymerization. Studies on immobilization of Ti or Sb on Ti-MMT or Sb-MMT are seldom reported. In this work, a series of montmorillonite supported catalysts of titanium (Ti-MMT) or antimony (Sb-MMT) and co-intercalated montmorillonite of titanium and antimony (Ti/Sb-MMT) were prepared by (1) the reaction of sodium bentonite suspension with intercalating solution containing titanium tetrachloride and/or antimony chloride, and (2) drying or calcinating the products at different temperature (100, 150, 240, 350 and 450℃). The physicochemical properties of these MMT supported catalysts were studied by X-ray diffraction (XRD), fourier transform infrared spectroscopy (FT-IR), inductively coupled plasma optical emission spectrometer (ICPOES), N2 adsorption/desorption isotherms, UV-visible diffuse reflectance spectroscopy(UV-vis) and transmission electron microscopy (TEM). The immobile character of Ti or Sb on MMT supported catalysts was evaluated by a two-step method in deionized water or ethylene glycol. Several results were obtained, i e, (a) during the preparation, with an increase in drying or calcinating temperature, the amount of titanium and/or antimony species remained on these MMT supported catalysts decreased, (b) the experiments about immobile character of Ti or/and Sb showed that with an increase in drying or calcinating temperature, the immobilization of Ti and/or Sb species remained on these MMT supported catalysts increased gradually, (c) Ti-MMT calcinated at 450℃had the biggest pore volume, which means Ti-MMT had the best adsorption application prospect.

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.

Science.gov (United States)

Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita; Chaudhary, Vijay K

2018-01-01

The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.

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.

Immobilization of Na,K-ATPase isolated from rat brain synaptic plasma membranes

Directory of Open Access Journals (Sweden)

ANICA HROVAT

2002-12-01

Full Text Available Rat brain Na,K-ATPase partially purified by SDS from synaptic plasma membranes (SPM was immobilized by adsorption on nitrocellulose (NC, polyvinylidene fluoride (PVDF and glass fiber (GF membranes. Partial SDS solubilization increased the enzyme activity by 40 %. With regard to the preservation of the enzyme activity, nitrocellulose was shown to be the optimal support for the immobilization. The enzyme showed the highest percentage activity (14 % after 30 min of SPM adsorption, at 20°C under the vaccum, with 25 mg of proteins per NC disc filter. In addition, adsorption on NC stabilizes the Na,K-ATPase, since the activity was substantial 72 h after adsorption at 20°C. After adsorption, the sensitivity of the enzyme to HgCl2and CdCll2 inhibition was higher. The results show that immobilized Na,K-ATPase SPM can be used as a practical model for the detection of metal ions in different samples.

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)

The characterization and testing of candidate immobilization forms for the disposal of plutonium

International Nuclear Information System (INIS)

Bakel, A. J.; Buck, E. C.; Chamberlain, D. B.; Ebbinghaus, B. B.; Fortner, J. A.; Marra, J. C.; Mcgrail, B. P.; Mertz, C. J.; Peeler, D. K.; Shaw, H. F.; Strachan, D. M.; Van Konynenburg, R. A.; Vienna, J. D.; Wolf, S. F.

1997-01-01

Candidate immobilization forms for the disposal of surplus weapons-useable are being tested and characterized. The goal of the testing program was to provide sufficient data that, by August 1997, an informed selection of a single immobilization form could be made so that the form development and production R and D could be more narrowly focused. Two forms have been under consideration for the past two years: glass and ceramic. In August, 1997, the Department of Energy (DOE) selected ceramic for plutonium disposition, halting further work on the glass material. In this paper, we will briefly describe these two waste forms, then describe our characterization techniques and testing methods. The analytical methods used to characterize altered and unaltered samples are the same. A full suite of microscopic techniques is used. Techniques used include optical, scanning electron, and transmission electron microscopies. For both candidate immobilization forms, the analyses are used to characterize the material for the presence of crystalline phases and amorphous material. Crystalline materials, either in the untested immobilization form or in the alteration products from testing, are characterized with respect to morphology, crystal structure, and composition. The goal of these analyses is to provide data on critical issues such as Pu and neutron absorber volubility in the immobilization form, thermal stability, potential separation of absorber and Pu, and the long-term behavior of the materials. Results from these analyses will be discussed in the presentation. Testing methods include MCC-1 tests, product consistency tests (methods A and B), unsaturated ''drip'' tests, vapor hydration tests, single-pass flow-through tests, and pressurized unsaturated flow tests. Both candidate immobilization forms have very low dissolution rates; examples of typical test results will be reported

Optically and biologically active mussel protein-coated double-walled carbon nanotubes.

Science.gov (United States)

Jung, Yong Chae; Muramatsu, Hiroyuki; Fujisawa, Kazunori; Kim, Jin Hee; Hayashi, Takuya; Kim, Yoong Ahm; Endo, Morinobu; Terrones, Mauricio; Dresselhaus, Mildred S

2011-12-02

A method of dispersing strongly bundled double-walled carbon nanotubes (DWNTs) via a homogeneous coating of mussel protein in an aqueous solution is presented. Optical activity, mechanical strength, as well as electrical conductivity coming from the nanotubes and the versatile biological activity from the mussel protein make mussel-coated DWNTs promising as a multifunctional scaffold and for anti-fouling materials. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optical tweezers reveal how proteins alter replication

Science.gov (United States)

Chaurasiya, Kathy

Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic

Determination of Concentration of Living Immobilized Yeast Cells by Fluorescence Spectroscopy

Czech Academy of Sciences Publication Activity Database

Podrazký, Ondřej; Kuncová, Gabriela

2005-01-01

Roč. 107, č. 1 (2005), s. 126-134 ISSN 0925-4005. [European Conference on Optical Chemical Sensors and Biosensors EUROPT(R)ODE /7./. Madrid, 04.04.2004-07.04.2004] R&D Projects: GA ČR GA104/01/0461; GA MŠk(CZ) OC 840.10 Institutional research plan: CEZ:AV0Z40720504 Keywords : immobilization of cells * 2-D fluorescence spectroscopy * sol–gel Subject RIV: CE - Biochemistry Impact factor: 2.646, year: 2005

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.

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...

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.

New developments for the site-specific attachment of protein to surfaces

Energy Technology Data Exchange (ETDEWEB)

Camarero, J A

2005-05-12

Protein immobilization on surfaces is of great importance in numerous applications in biology and biophysics. The key for the success of all these applications relies on the immobilization technique employed to attach the protein to the corresponding surface. Protein immobilization can be based on covalent or noncovalent interaction of the molecule with the surface. Noncovalent interactions include hydrophobic interactions, hydrogen bonding, van der Waals forces, electrostatic forces, or physical adsorption. However, since these interactions are weak, the molecules can get denatured or dislodged, thus causing loss of signal. They also result in random attachment of the protein to the surface. Site-specific covalent attachment of proteins onto surfaces, on the other hand, leads to molecules being arranged in a definite, orderly fashion and uses spacers and linkers to help minimize steric hindrances between the protein surface. This work reviews in detail some of the methods most commonly used as well as the latest developments for the site-specific covalent attachment of protein to solid surfaces.

Immobilization of Highly Effective Palladium Catalyst onto Poly(4-Vinylpyridine): Synthesis and Characterization

International Nuclear Information System (INIS)

Siti Kamilah Che Soh; Intan Shafinass Kassim; Siti Aminah Jusoh; Mustaffa Samsuddin

2016-01-01

A commonly known weakness of homogeneous catalysts is the difficulty to recover the active catalyst from the product. Due to the disadvantage, the designing of supported catalyst has been approached to overcome the separation difficulty of the palladium-based homogeneous catalyst. New polymer supported N 2 O 2 metal complex was successfully immobilized by mixing of poly(4-vinylpyridine) with palladium(II) complex in the presence of ethyl acetate as solvent. Then, the reaction was stirred for 72 hours at room temperature to form corresponding P 4 VP-Pd catalyst. The properties of immobilized catalyst were characterized by various techniques such as fourier transform infrared (FTIR), thermogravimetric (TGA), X-ray diffraction (XRD), scanning electron microscopy/ energy dispersive X-ray (SEM/ EDX) and inductively coupled plasma-optical emission (ICP-OES) spectroscopy. (author)

Multiplex single-molecule interaction profiling of DNA barcoded proteins

Science.gov (United States)

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

2014-01-01

In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

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.

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.

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.

A CORN STEM AS BIOMATERIAL FOR SACCHAROMYCES CEREVISIAE CELLS IMMOBILIZATION FOR THE ETHANOL PRODUCTION

Directory of Open Access Journals (Sweden)

Vesna Vučurović

2008-11-01

Full Text Available This study provides a preliminary contribution to the development of a bioprocess for the production of ethanol using Saccharomyces cerevisiae cells immobilized onto a corn stem. For this purpose, the yeast cells were submitted to the batch tests in situ adsorption onto 0.5 cm long corn stem. Cells immobilization was analyzed by optical microscopy. The number of the yeast cells, fermentation kinetics, the ethanol yield in the presence or the absence of the support in the fermentation medium was investigated. It was determined that the addition of the corn stem led to the abrupt increase of the yeast cells number in substrate, ethanol yield, pH value, a total dissolved salts content and substrate conductivity. The addition of 5 and 10g of the corn stem pith per liter of the medium decreased the amount of residual sugar. The results indicate that a corn stem might be a good carrier for the yeast cell immobilization, and also a cheap alternative recourse of mineral components with the possibility of application for improving ethanol productivities.

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.

Energy transfer between surface-immobilized light-harvesting chlorophyll a/b complex (LHCII) studied by surface plasmon field-enhanced fluorescence spectroscopy (SPFS).

Science.gov (United States)

Lauterbach, Rolf; Liu, Jing; Knoll, Wolfgang; Paulsen, Harald

2010-11-16

The major light-harvesting chlorophyll a/b complex (LHCII) of the photosynthetic apparatus in green plants can be viewed as a protein scaffold binding and positioning a large number of pigment molecules that combines rapid and efficient excitation energy transfer with effective protection of its pigments from photobleaching. These properties make LHCII potentially interesting as a light harvester (or a model thereof) in photoelectronic applications. Most of such applications would require the LHCII to be immobilized on a solid surface. In a previous study we showed the immobilization of recombinant LHCII on functionalized gold surfaces via a 6-histidine tag (His tag) in the protein moiety. In this work the occurrence and efficiency of Förster energy transfer between immobilized LHCII on a functionalized surface have been analyzed by surface plasmon field-enhanced fluorescence spectroscopy (SPFS). A near-infrared dye was attached to some but not all of the LHC complexes, serving as an energy acceptor to chlorophylls. Analysis of the energy transfer from chlorophylls to this acceptor dye yielded information about the extent of intercomplex energy transfer between immobilized LHCII.

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 ®}.

Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.

Science.gov (United States)

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-08-01

INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.

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

Production of immobilized cellulase enzyme by some microorganisms from the rice straw agro-waste using γ-irradiation

International Nuclear Information System (INIS)

Mohamed, M.A.Z.

2014-01-01

Studies were carried out using 14 fungal cultures screened for their ability to produce cellulase enzymes. A .hortai was selected for the present research as a potent cellulase producer. Cultural and nutritional factors affecting cellulase production were also investigated in order to optimize the fermentation conditions for the maximization of production. The obtained results revealed that, the maximum cellulase production (0.23 U/ml) was achieved after 96 h in a liquid medium (Ph 7.0) inoculated with 10% v/v inoculum size, at temperature 37 ºC, containing (gL -1 ) CMC, 5.0; yeast extract, 0.1; (NH 4 ) 2 SO 4 , 0.5; KH 2 PO 4 , 10.0; MgSO 4 .7H 2 O, 0.1 and NaCl, 0.2. The activity remained almost stable between ph 6.0 and 7.0. The highest cellulase activity (1.18 U/ml) was obtained at a lactose concentration of (5.0 gL -1 ). Partial purification of the crude cellulase by ammonium sulphate 70% saturation showed the highest specific enzyme activity and purification fold (2.3 U/mg protein and 2.12 fold, respectively). Different carriers and methods were used to select the suitable one for cellulase immobilization. Poly (acrylamide-co-acrylic acid) prepared by diazotization method increase S.E.A and the amount of immobilized enzyme to be (2.3 U/mg protein and 2.8 mg), respectively. The immobilized cellulase shows better operational stability, including wider ph and thermal ranges. The immobilized cellulase remained fully active up to 60°C. The kinetic parameters Km and Vmax were determined. The increase of the apparent Km after immobilization clearly indicates an apparent lower affinity of the immobilized enzyme for its substrate than the free enzyme. The resulting immobilized cellulase exhibited good reusability on degradation of rice straw agricultural wastes and also show good storage stability, that it lost only 17 % of its initial activity after 6 weeks.

Laser-based optical activity detection of amino acids and proteins

Energy Technology Data Exchange (ETDEWEB)

Reitsma, B.H.

1987-01-01

The optical activity detector (OAD) for HPLC is a selective detector for optically active substances including amino acids and proteins. This study illustrates the use of the OAD in three related areas. Section I illustrates the separation of four free amino acids using cation-exchange chromatography. Detection by coupling the OAD to a refractive index detector (RI) for proline and threonine and the OAD to an ultraviolet absorbance detector (UV) for tyrosine and phenylalanine allows the calculation of enantiomeric (D/L) ratios of these amino acids without physical separation. Specific rotations of these four amino acids are also reported. Section II illustrates the separation of 16 dansyl-L-amino acids by RP-HPLC with detection by OA/UV. Section III illustrates the RP-HPLC separation of conformers of soybean trypsin inhibitor. Detection by OA/UV provides insights from the chromatogram unavailable for UV absorbance detection alone. In addition, identification of impurities is simplified with OA/UV. Specific rotations of the separated protein fractions show no significant change accompanying change in conformation.

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.

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

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

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

Laser-optical investigation of the effect of diamond nanoparticles on the structure and functional properties of proteins

International Nuclear Information System (INIS)

Perevedentseva, Elena V; Su, F.Y.; Su, T.H.; Lin, Y.C.; Cheng, C.L.; Karmenyan, A V; Priezzhev, A V; Lugovtsov, Andrei E

2011-01-01

Adsorption of such blood plasma proteins as albumin and g-globulin on diamond nanoparticles of size around 5 nm and around 100 nm is observed and studied using laser-optical methods. The adsorption of blood plasma proteins at physiological pH 7.4 is found weaker than that of enzyme protein lysozyme. The observed variations in the Fourier Transform Infrared (FTIR) spectra of proteins may be due to structural transformations of the adsorbed protein. Using the lysozyme as a test protein we show that the protein adsorption leading to observable changes in the FTIR spectrum (the band of Amide I) also induces a significant decrease in the protein functional activity. It is also found that the influence of ∼5-nm diamond nanoparticles on the protein structure and functions is more significant than that of ∼100-nm nanodiamonds. (application of lasers and laser-optical methods in life sciences)

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.

Immobilization Patterns and Dynamics of Acetate-Utilizing Methanogens Immobilized in Sterile Granular Sludge in Upflow Anaerobic Sludge Blanket Reactors

Science.gov (United States)

Schmidt, Jens Ejbye; Ahring, Birgitte Kjær

1999-01-01

Sterile granular sludge was inoculated with either Methanosarcina mazeii S-6, Methanosaeta concilii GP-6, or both species in acetate-fed upflow anaerobic sludge blanket (UASB) reactors to investigate the immobilization patterns and dynamics of aceticlastic methanogens in granular sludge. After several months of reactor operation, the methanogens were immobilized, either separately or together. The fastest immobilization was observed in the reactor containing M. mazeii S-6. The highest effluent concentration of acetate was observed in the reactor with only M. mazeii S-6 immobilized, while the lowest effluent concentration of acetate was observed in the reactor where both types of methanogens were immobilized together. No changes were observed in the kinetic parameters (Ks and μmax) of immobilized M. concilii GP-6 or M. mazeii S-6 compared with suspended cultures, indicating that immobilization does not affect the growth kinetics of these methanogens. An enzyme-linked immunosorbent assay using polyclonal antibodies against either M. concilii GP-6 or M. mazeii S-6 showed significant variations in the two methanogenic populations in the different reactors. Polyclonal antibodies were further used to study the spatial distribution of the two methanogens. M. concilii GP-6 was immobilized only on existing support material without any specific pattern. M. mazeii S-6, however, showed a different immobilization pattern: large clumps were formed when the concentration of acetate was high, but where the acetate concentration was low this strain was immobilized on support material as single cells or small clumps. The data clearly show that the two aceticlastic methanogens immobilize differently in UASB systems, depending on the conditions found throughout the UASB reactor. PMID:10049862

Advanced optical measurements for characterizing photophysical properties of single nanoparticles.

Energy Technology Data Exchange (ETDEWEB)

Polsky, Ronen; Davis, Ryan W.; Arango, Dulce C.; Brozik, Susan Marie; Wheeler, David Roger

2009-09-01

Formation of complex nanomaterials would ideally involve single-pot reaction conditions with one reactive site per nanoparticle, resulting in a high yield of incrementally modified or oriented structures. Many studies in nanoparticle functionalization have sought to generate highly uniform nanoparticles with tailorable surface chemistry necessary to produce such conjugates, with limited success. In order to overcome these limitations, we have modified commercially available nanoparticles with multiple potential reaction sites for conjugation with single ssDNAs, proteins, and small unilamellar vesicles. These approaches combined heterobifunctional and biochemical template chemistries with single molecule optical methods for improved control of nanomaterial functionalization. Several interesting analytical results have been achieved by leveraging techniques unique to SNL, and provide multiple paths for future improvements for multiplex nanoparticle synthesis and characterization. Hyperspectral imaging has proven especially useful for assaying substrate immobilized fluorescent particles. In dynamic environments, temporal correlation spectroscopies have been employed for tracking changes in diffusion/hydrodynamic radii, particle size distributions, and identifying mobile versus immobile sample fractions at unbounded dilution. Finally, Raman fingerprinting of biological conjugates has been enabled by resonant signal enhancement provided by intimate interactions with nanoparticles and composite nanoshells.

Investigation of HIV-1 infected and uninfected cells using the optical trapping technique

CSIR Research Space (South Africa)

Ombinda-Lemboumba, Saturnin

2017-02-01

Full Text Available Optical trapping has emerged as an essential tool for manipulating single biological material and performing sophisticated spectroscopy analysis on individual cell. The optical trapping technique has been used to grab and immobilize cells from a...

Plasma Treated High-Density Polyethylene (HDPE Medpor Implant Immobilized with rhBMP-2 for Improving the Bone Regeneration

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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.

Highly sensitive optical sensor that detects Hg{sup 2+} and Cu{sup 2+} by immobilizing dicarboxylate 1,5-diphenyl-3-thiocarbazone on surface functionalized PVA microspheres

Energy Technology Data Exchange (ETDEWEB)

Bai, Xue, E-mail: baixue@hhu.edu.cn; Gu, Haixin; Hua, Zulin; Dai, Zhangyan; Yang, Bei; Li, Yulong

2015-11-15

Highlights: • PVA microspheres were chosen as carrier and DDT groups were chosen as chromophores. • The DDT–PVA microspheres could detect Hg{sup 2+} and Cu{sup 2+} simultaneously within 120 s. • The DDT–PVA microspheres had excellent detection for Hg{sup 2+} and Cu{sup 2+} ions. • The DDT–PVA microspheres had preeminent selectivity and reusability. - Abstract: A novel optical sensor to detect Hg{sup 2+} and Cu{sup 2+} is prepared by immobilizing a synthesized dicarboxylate 1,5-diphenyl-3-thiocarbazone (DDT) group on functionalized polyvinyl alcohol (PVA) microspheres. This optical sensor is successfully fabricated by extensive characterization with Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Its colorimetric properties, selectivity, sensitivity, and reversibility are investigated as well. In this sensing system, DDT–PVA selectively recognized multiple heavy metal ions, as indicated by the changes in color from orange to scarlet for Hg{sup 2+} and from orange to gray for Cu{sup 2+}. In particular, this optical sensor exhibits the most apparent color changes at pH levels of 12 and 2. Hence, Hg{sup 2+} and Cu{sup 2+} can be detected in aqueous solution at minimum detection limits of 0.053 and 0.132 μM, respectively, with a UV-vis spectrometer. Furthermore, the sensor can be regenerated by ethylene diamine tetraacetic acid and reused several times. Therefore, the optical sensor can detect Hg{sup 2+} because of its selectivity, sensitivity, and reversibility.

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.

Immobilization of β-glucosidase onto mesoporous silica support: Physical adsorption and covalent binding of enzyme

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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

Membrane Compartmentalization Reducing the Mobility of Lipids and Proteins within a Model Plasma Membrane.

Science.gov (United States)

Koldsø, Heidi; Reddy, Tyler; Fowler, Philip W; Duncan, Anna L; Sansom, Mark S P

2016-09-01

The cytoskeleton underlying cell membranes may influence the dynamic organization of proteins and lipids within the bilayer by immobilizing certain transmembrane (TM) proteins and forming corrals within the membrane. Here, we present coarse-grained resolution simulations of a biologically realistic membrane model of asymmetrically organized lipids and TM proteins. We determine the effects of a model of cytoskeletal immobilization of selected membrane proteins using long time scale coarse-grained molecular dynamics simulations. By introducing compartments with varying degrees of restraints within the membrane models, we are able to reveal how compartmentalization caused by cytoskeletal immobilization leads to reduced and anomalous diffusional mobility of both proteins and lipids. This in turn results in a reduced rate of protein dimerization within the membrane and of hopping of membrane proteins between compartments. These simulations provide a molecular realization of hierarchical models often invoked to explain single-molecule imaging studies of membrane proteins.

Immobilized/P25/DSAT and Immobilized/Kronos/DSAT on Photocatalytic Degradation of Reactive Red 4 Under Fluorescent Light

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Azami M. S.

2016-01-01

Full Text Available In this work, photocatalytic degradation of Reactive Red 4 (RR4 using immobilized P25 and kronos were performed under fluorescent light sources. The photocatalysis activity for both catalysts was investigated under fluorescent lamp source which consist UV and Visible light. The effect of various parameters such as initial concentration, initial pH and strenght of immobilized plate were studied. The result showed that 90% of RR4 dye was degrade in 1 hr using immobilized/kronos/DSAT at 100 mg L-1 of RR4 dye while 81% degradation was achieved by immobilized/P25/DSAT at the same condition. The lowest pH showed the higher photocatalytic activity. Hence, the effect of dye concentration and pH on the photocatalysis study can be related with the behavior of environmental pollution. The low strength showed by immobilized/P25/DSAT where it remain 37 % as compared with strength of immobilized/kronos/DSAT (52 wt.%. For the future work, the polymer binder like Polyvinyl alcohol (PVA, Polyethylene glycol (PEG, and others polymers can be apply in immobilized study to overcome the strength problem.

Evaluation of inorganic matrixes as supports for immobilization of microbial lipase

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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.

Scanning a DNA molecule for bound proteins using hybrid magnetic and optical tweezers.

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Marijn T J van Loenhout

Full Text Available The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33 ± 5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/[Formula: see text]. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.

Halloysite nanotubes with immobilized silver nanoparticles for anti-bacterial application.

Science.gov (United States)

Jana, Subhra; Kondakova, Anastasiya V; Shevchenko, Svetlana N; Sheval, Eugene V; Gonchar, Kirill A; Timoshenko, Victor Yu; Vasiliev, Alexander N

2017-03-01

Halloysite nanotubes (HNTs) with immobilized silver (Ag) nanoparticles (NPs) were prepared by methods of wet chemistry and were characterized by using the transmission electron microscopy, x-ray diffraction, optical spectroscopy and experiments with E. coli bacteria in-vitro. It was found that Ag NPs with almost perfect crystalline structure and sizes from ∼9nm were mainly attached over the external surface of HNTs. The optical absorption measurement revealed a broad plasmonic resonance in the region of 400-600nm for HNTs with Ag NPs. The later samples exhibit bactericidal effect, which is more pronounced under illumination. A role of the plasmonic excitation of Ag NPs for their bioactive properties is discussed. The obtained results show that Ag NPs-decorated HNTs are promising agents for the antibacterial treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

Design-Only Conceptual Design Report: Plutonium Immobilization Plant

International Nuclear Information System (INIS)

DiSabatino, A.; Loftus, D.

1999-01-01

This design-only conceptual design report was prepared to support a funding request by the Department of Energy Office of Fissile Materials Disposition for engineering and design of the Plutonium Immobilization Plant, which will be used to immobilize up to 50 tonnes of surplus plutonium. The siting for the Plutonium Immobilization Plant will be determined pursuant to the site-specific Surplus Plutonium Disposition Environmental Impact Statement in a Plutonium Deposition Record of Decision in early 1999. This document reflects a new facility using the preferred technology (ceramic immobilization using the can-in-canister approach) and the preferred site (at Savannah River). The Plutonium Immobilization Plant accepts plutonium from pit conversion and from non-pit sources and, through a ceramic immobilization process, converts the plutonium into mineral-like forms that are subsequently encapsulated within a large canister of high-level waste glass. The final immobilized product must make the plutonium as inherently unattractive and inaccessible for use in nuclear weapons as the plutonium in spent fuel from commercial reactors and must be suitable for geologic disposal. Plutonium immobilization at the Savannah River Site uses: (1) A new building, the Plutonium Immobilization Plant, which will convert non-pit surplus plutonium to an oxide form suitable for the immobilization process, immobilize plutonium in a titanate-based ceramic form, place cans of the plutonium-ceramic forms into magazines, and load the magazines into a canister; (2) The existing Defense Waste Processing Facility for the pouring of high-level waste glass into the canisters; and (3) The Actinide Packaging and Storage Facility to receive and store feed materials. The Plutonium Immobilization Plant uses existing Savannah River Site infra-structure for analytical laboratory services, waste handling, fire protection, training, and other support utilities and services. The Plutonium Immobilization Plant

Radiation target analyses of free and immobilized glucose 6-phosphate dehydrogenase

International Nuclear Information System (INIS)

Kempner, E.S.; Miller, J.H.

2010-01-01

The sensitivity of the enzyme glucose 6-phosphate dehydrogenase to ionizing radiation was examined under several conditions, including the presence of several free-radical scavengers. The enzyme was also irradiated when covalently bound to polyacrylamide beads whose structure is very similar to the polypeptide backbone of proteins. All the enzyme forms were irradiated in the frozen state with high-energy electrons from a linear accelerator. Surviving enzyme activity and surviving monomers were determined; the data were analyzed by target theory. Free-radical scavengers reduced the radiation target size of both the activity and monomers of the free enzyme, but not that of the immobilized enzyme activity. The target size of the activity of the free enzyme was that of a dimer mass, but in the case of the immobilized enzyme it was equal to the smaller mass of the monomer. Free-radical scavengers reduce the target size by modifying radiation energy transfer. The target size of the polyacrylamide-bound enzyme activity was expected to be very large since the connection between polyacrylamide and protein is a peptide bond which permits transfer of radiation-deposited energy. Several explanations concerning energy transfer are suggested for this result.

Formation of protein-birnessite complex: XRD, FTIR, and AFM analysis.

Science.gov (United States)

Naidja, A; Liu, C; Huang, P M

2002-07-01

Limited information is available on formation chemistry of enzyme-Mn oxide complexes. Adsorption isotherm of protein molecules (tyrosinase) on birnessite (delta-MnO(2)) at pH 6.0 and room temperature (23 degrees C) was of H type, indicating a very high affinity of the enzyme protein molecules to the birnessite mineral surfaces. After thorough washing of the protein-mineral complex with deionized-distilled water, up to 89% of adsorbed protein molecules remained bound to the mineral surfaces. When a high amount of the protein was immobilized, the X-ray diffractogram shows a significant decrease in the intensity of characteristic d-spacings of birnessite. No shift to higher values of the d-spacings of protein-birnessite complex was observed, indicating that the enzyme molecules were not intercalated in the mineral structure but immobilized at the external surfaces and the edges of the mineral oxide. By comparison to the free enzyme, infrared absorption spectra of the protein-birnessite complexes show a shift by up to 11 cm(-1) to lower frequencies in the absorption bands characteristic of amide I and II modes of the polypeptides chains. The mineral surfaces exerted some strain on the protein structure, resulting in an alteration of the protein molecular conformation after binding to the mineral colloid surfaces. In the free state, the globular protein molecules had a spheroid shape with an average cross-sectional diameter of 70+/-6 nm. The unfolding and flattening of the protein molecules after immobilization is clearly shown in atomic force micrographs. Compared to the tyrosinase-birnessite complex, similar FTIR spectra and atomic force micrographs were observed for the pure protein, bovine serum albumin (BSA), after immobilization on birnessite. The information obtained in this study is of fundamental significance for understanding birnessite as an adsorbent of biopolymers and the catalytic role of the enzyme-birnessite complex.

Lipid-mediated protein functionalization of electrospun polycaprolactone fibers

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C. Cohn

2016-05-01

Full Text Available In this study, electrospun polycaprolactone (PCL fibers are plasma-treated and chemically conjugated with cholesteryl succinyl silane (CSS. In addition to Raman spectroscopy, an immobilization study of DiO as a fluorescent probe of lipid membranes provides evidence supporting the CSS coating of plasma-treated PCL fibers. Further, anti-CD20 antibodies are used as a model protein to evaluate the potential of lipid-mediated protein immobilization as a mechanism to functionalize the CSS-PCL fiber scaffolds. Upon anti-CD20 functionalization, the CSS-PCL fiber scaffolds capture Granta-22 cells 2.4 times more than the PCL control does, although the two fiber scaffolds immobilize a comparable amount of anti-CD20. Taken together, results from the present study demonstrate that the CSS coating and CSS-mediated antibody immobilization offers an appealing strategy to functionalize electrospun synthetic polymer fibers and confer cell-specific functions on the fiber scaffolds, which can be mechanically robust but often lack biological functions.

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.

Continuous-flow enantioselective α-aminoxylation of aldehydes catalyzed by a polystyrene-immobilized hydroxyproline

Directory of Open Access Journals (Sweden)

Xacobe C. Cambeiro

2011-10-01

Full Text Available The application of polystyrene-immobilized proline-based catalysts in packed-bed reactors for the continuous-flow, direct, enantioselective α-aminoxylation of aldehydes is described. The system allows the easy preparation of a series of β-aminoxy alcohols (after a reductive workup with excellent optical purity and with an effective catalyst loading of ca. 2.5% (four-fold reduction compared to the batch process working at residence times of ca. 5 min.

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

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)

Rapid and efficient proteolysis through laser-assisted immobilized enzyme reactors.

Science.gov (United States)

Zhang, Peng; Gao, Mingxia; Zhu, Shaochun; Lei, Jie; Zhang, Xiangmin

2011-11-25

In this report, laser radiation (808nm) for the first time was employed to enhance the efficiency of proteolysis through immobilized enzyme reactor (IMER). IMER based monolithic support was prepared in the fused-silica capillary via a simple two-step procedure including acryloylation on trypsin surface and in situ aqueous polymerization/immobilization. The feasibility and high efficiency of the laser-assisted IMER were demonstrated by the digestion of bovine serum albumin (BSA), cytochrome c (Cyt-c) and β-casein. The digestion process was achieved in 60s. The peptides were identified by MALDI-TOF-MS, yielding the sequence coverage of 33% for BSA, 73% for Cyt-c and 22% for β-casein. The comparisons between the in-solution digestion and on IMER reaction with/without laser assistance were made. To further confirm its efficiency in proteome analysis, the laser-assisted IMER was also applied to the analysis of one fraction of human serum sample through two-dimensional (2-D) separation of strong anion exchange/reversed-phase liquid chromatography (SAX/RPLC). After a database search, 49 unique peptides corresponding to 5 proteins were identified. The results showed that the laser-assisted IMER provides a promising platform for the high-throughput protein identification. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

Synthesis and Characterization of PEDOT Derivative with Carboxyl Group and Its Chemo/Bio Sensing Application as Nanocomposite, Immobilized Biological and Enhanced Optical Materials

International Nuclear Information System (INIS)

Zhang, Long; Wen, Yangping; Yao, Yuanyuan; Xu, Jingkun; Duan, Xuemin; Zhang, Ge

2014-01-01

indicate that chemo/bio-sensors based on PC4 possess excellent chemo/bio sensing performance and enhanced optical response, and its carboxylic group can realize the immobilization of biologically active species, self-assembly of inorganic nanomaterials

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)

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.)

Characteristics of Immobilized Urease on Grafted Alginate Bead Systems

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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.

Larger red-shift in optical emissions obtained from the thin films of globular proteins (BSA, lysozyme) – polyelectrolyte (PAA) complexes

Energy Technology Data Exchange (ETDEWEB)

Talukdar, Hrishikesh [Physical Sciences Division, Institute of Advanced Study in Science and Technology, Vigyan Path, Paschim Boragaon, Garchuk, Guwahati 781035, Assam (India); Kundu, Sarathi, E-mail: sarathi.kundu@gmail.com [Physical Sciences Division, Institute of Advanced Study in Science and Technology, Vigyan Path, Paschim Boragaon, Garchuk, Guwahati 781035, Assam (India); Basu, Saibal [Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India)

2016-09-30

Graphical abstract: Thin films of protein-polyelectrolyte complexes show larger red-shift in optical emission. - Highlights: • Globular proteins (lysozyme and BSA) and polyelectrolyte (sodium polyacrylic acid) are used to form protein-polyelectrolyte complexes (PPC). • Larger red-shift in optical emission is obtained from the thin films of PPC. • Red-shift is not obtained from the solution of PPC and pure protein thin films. • Larger red-shift from PPC films is due to the energy dissipation as non-radiative form through interactions with nearby atoms. • Red-shift in optical emission is independent on the thickness of the PPC film. - Abstract: Globular proteins (lysozyme and BSA) and polyelectrolyte (sodium polyacrylic acid) are used to form protein-polyelectrolyte complexes (PPC). Out-of-plane structures of ≈30–60 nm thick PPC films and their surface morphologies have been studied by using X-ray reflectivity and atomic force microscopy, whereas optical behaviors of PPC and protein conformations have been studied by using UV–vis, photoluminescence and FTIR spectroscopy respectively. Our study reveals that thin films of PPC show a larger red-shift of 23 and 16 nm in the optical emissions in comparison to that of pure protein whereas bulk PPC show a small blue-shift of ≈3 nm. A small amount of peak-shift is found to occur due to the heat treatment or concentration variation of the polyelectrolyte/protein in bulk solution but cannot produce such film thickness independent larger red-shift. Position of the emission peak remains nearly unchanged with the film thickness. Mechanism for such larger red-shift has been proposed.

DNA origami nanorobot fiber optic genosensor to TMV.

Science.gov (United States)

Torelli, Emanuela; Manzano, Marisa; Srivastava, Sachin K; Marks, Robert S

2018-01-15

In the quest of greater sensitivity and specificity of diagnostic systems, one continually searches for alternative DNA hybridization methods, enabling greater versatility and where possible field-enabled detection of target analytes. We present, herein, a hybrid molecular self-assembled scaffolded DNA origami entity, intimately immobilized via capture probes linked to aminopropyltriethoxysilane, onto a glass optical fiber end-face transducer, thus producing a novel biosensor. Immobilized DNA nanorobots with a switchable flap can then be actuated by a specific target DNA present in a sample, by exposing a hemin/G-quadruplex DNAzyme, which then catalyzes the generation of chemiluminescence, once the specific fiber probes are immersed in a luminol-based solution. Integrating organic nanorobots to inorganic fiber optics creates a hybrid system that we demonstrate as a proof-of-principle can be utilized in specific DNA sequence detection. This system has potential applications in a wide range of fields, including point-of-care diagnostics or cellular in vivo biosensing when using ultrathin fiber optic probes for research purposes. Copyright © 2017 Elsevier B.V. All rights reserved.

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 ...

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.

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

Specific capture of uranyl protein targets by metal affinity chromatography

International Nuclear Information System (INIS)

Basset, C.; Dedieu, A.; Guerin, P.; Quemeneur, E.; Meyer, D.; Vidaud, C.

2008-01-01

To improve general understanding of biochemical mechanisms in the field of uranium toxicology, the identification of protein targets needs to be intensified. Immobilized metal affinity chromatography (IMAC) has been widely developed as a powerful tool for capturing metal binding proteins from biological extracts. However uranyl cations (UO 2 2+ ) have particular physico-chemical characteristics which prevent them from being immobilized on classical metal chelating supports. We report here on the first development of an immobilized uranyl affinity chromatography method, based on the cation-exchange properties of amino-phosphonate groups for uranyl binding. The cation distribution coefficient and loading capacity on the support were determined. Then the stability of the uranyl-bonded phase under our chromatographic conditions was optimized to promote affinity mechanisms. The successful enrichment of uranyl binding proteins from human serum was then proven using proteomic and mass spectral analysis. (authors)

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

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.

Immobilization of Murine Anti-BMP-2 Monoclonal Antibody on Various Biomaterials for Bone Tissue Engineering

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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.

Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats

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L. Gavrilovic

2009-12-01

Full Text Available Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH, dopamine-β-hydroxylase (DBH and phenylethanolamine N-methyltransferase (PNMT and protein levels in the right and left heart auricles of naive control and long-term (12 weeks socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70% compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62% and left (about 81% auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267%, DBH (about 37% and PNMT (about 60% only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.

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.

The utilization of BSA-modified chip on the investigation of ligand ...

African Journals Online (AJOL)

Administrator

2009-12-15

Dec 15, 2009 ... investigation of ligand/protein interaction with surface plasma resonance ... for immobilizing proteins or low-molecular-weight ligands to dextran ..... contamination in dynamic aqueous environments using optical sensors. Anal.

Optical Fiber Sensor Based on Localized Surface Plasmon Resonance Using Silver Nanoparticles Photodeposited on the Optical Fiber End

Directory of Open Access Journals (Sweden)

J. Gabriel Ortega-Mendoza

2014-10-01

Full Text Available This paper reports the implementation of an optical fiber sensor to measure the refractive index in aqueous media based on localized surface plasmon resonance (LSPR. We have used a novel technique known as photodeposition to immobilize silver nanoparticles on the optical fiber end. This technique has a simple instrumentation, involves laser light via an optical fiber and silver nanoparticles suspended in an aqueous medium. The optical sensor was assembled using a tungsten lamp as white light, a spectrometer, and an optical fiber with silver nanoparticles. The response of this sensor is such that the LSPR peak wavelength is linearly shifted to longer wavelengths as the refractive index is increased, showing a sensitivity of 67.6 nm/RIU. Experimental results are presented.

Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope.

Science.gov (United States)

Fei, Yiyan; Landry, James P; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S

2010-01-01

We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm x 4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide.

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

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%.

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

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)

Design of a papain immobilized antimicrobial food package with curcumin as a crosslinker.

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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.

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

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.

Preparation of Laccase Immobilized Cryogels and Usage for Decolorization

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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.

Visualization of red-ox proteins on the gold surface using enzymatic polypyrrole formation

International Nuclear Information System (INIS)

Ramanaviciene, A.; Kausaite-Minkstimiene, A.; Voronovic, J.; Ramanavicius, A.; Oztekin, Y.; Carac, G.; German, N.

2011-01-01

We describe a new method for the visualization of the activity of red-ox proteins on a gold interface. Glucose oxidase was selected as a model system. Surfaces were modified by adhesion of glucose oxidase on (a) electrochemically cleaned gold; (b) gold films modified with gold nanoparticles, (c) a gold surface modified with self-assembled monolayer, and (d) covalent immobilization of protein on the gold surface modified with a self-assembled monolayer. The simple optical method for the visualization of enzyme on the surfaces is based on the enzymatic formation of polypyrrole. The activity of the enzyme was quantified via enzymatic formation of polypyrrole, which was detected and investigated by quartz microbalance and amperometric techniques. The experimental data suggest that the enzymatic formation of the polymer may serve as a method to indicate the adhesion of active redox enzyme on such surfaces. (author)

Ligand binding to G protein-coupled receptors in tethered cell membranes

DEFF Research Database (Denmark)

Martinez, Karen L.; Meyer, Bruno H.; Hovius, Ruud

2003-01-01

for the surface immobilization of membrane proteins was developed using the prototypic seven transmembrane neurokinin-1 receptor. The receptor was expressed as a biotinylated protein in mammalian cells. Membranes from cell homogenates were selectively immobilized on glass surfaces covered with streptavidin. TIRF...... measurements showed that a fluorescent agonist binds to the receptor on the sensor surface with similar affinity as to the receptor in live cells. This approach offers the possibility to investigate minute amounts of membrane protein in an active form and in its native environment without purification....

Molecular view of the interaction between iota-carrageenan and a phospholipid film and its role in enzyme immobilization.

Science.gov (United States)

Nobre, Thatyane M; de Sousa e Silva, Heurison; Furriel, Rosa P M; Leone, Francisco A; Miranda, Paulo B; Zaniquelli, Maria Elisabete D

2009-05-28

Proteins incorporated into phospholipid Langmuir-Blodgett (LB) films are a good model system for biomembranes and enzyme immobilization studies. The specific fluidity of biomembranes, an important requisite for enzymatic activity, is naturally controlled by varying phospholipid compositions. In a model system, instead, LB film fluidity may be varied by covering the top layer with different substances able to interact simultaneously with the phospholipid and the protein to be immobilized. In this study, we immobilized a carbohydrate rich Neurospora crassa alkaline phosphatase (NCAP) in monolayers of the sodium salt of dihexadecylphosphoric acid (DHP), a synthetic phospholipid that provides very condensed Langmuir films. The binding of NCAP to DHP Langmuir-Blodgett (LB) films was mediated by the anionic polysaccharide iota-carrageenan (iota-car). Combining results from surface isotherms and the quartz crystal microbalance technique, we concluded that the polysaccharide was essential to promote the interaction between DHP and NCAP and also to increase the fluidity of the film. An estimate of DHP:iota-car ratio within the film also revealed that the polysaccharide binds to DHP LB film in an extended conformation. Furthermore, the investigation of the polysaccharide conformation at molecular level, using sum-frequency vibrational spectroscopy (SFG), indicated a preferential conformation of the carrageenan molecules with the sulfate groups oriented toward the phospholipid monolayer, and both the hydroxyl and ether groups interacting preferentially with the protein. These results demonstrate how interfacial electric fields can reorient and induce conformational changes in macromolecules, which may significantly affect intermolecular interactions at interfaces. This detailed knowledge of the interaction mechanism between the enzyme and the LB film is relevant to design strategies for enzyme immobilization when orientation and fluidity properties of the film provided by the

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.

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

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

Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

Science.gov (United States)

Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

2016-03-01

DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are

Novel immobilization process of a thermophilic catalase: efficient purification by heat treatment and subsequent immobilization at high temperature.

Science.gov (United States)

Xu, Juan; Luo, Hui; López, Claudia; Xiao, Jing; Chang, Yanhong

2015-10-01

The main goal of the present work is to investigate a novel process of purification and immobilization of a thermophilic catalase at high temperatures. The catalase, originated from Bacillus sp., was overexpressed in a recombinant Escherichia coli BL21(DE3)/pET28-CATHis and efficiently purified by heat treatment, achieving a threefold purification. The purified catalase was then immobilized onto an epoxy support at different temperatures (25, 40, and 55 °C). The immobilizate obtained at higher temperatures reached its maximum activity in a shorter time than that obtained at lower temperatures. Furthermore, immobilization at higher temperatures required a lower ionic strength than immobilization at lower temperatures. The characteristics of immobilized enzymes prepared at different temperatures were investigated. The high-temperature immobilizate (55 °C) showed the highest thermal stability, followed by the 40 °C immobilizate. And the high-temperature immobilizate (55 °C) had slightly higher operational stability than the 25 °C immobilizate. All of the immobilized catalase preparations showed higher stability than the free enzyme at alkaline pH 10.0, while the alkali resistance of the 25 °C immobilizate was slightly better than that of the 40 and 55 °C immobilizates.

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.

Glucose Oxidase Immobilization on TMAH-Modified Bentonite

Directory of Open Access Journals (Sweden)

Ruth Chrisnasari

2015-03-01

Full Text Available The influence of bentonite modification by tetramethyl ammonium hydroxide (TMAH on its capability to immobilize glucose oxidase (GOX was studied. Modification of bentonite was conducted by the adding of 0-5% (v/v TMAH. The observed results show that the different concentrations of TMAH affect the percentage of immobilized enzyme. The results of this study show that the best concentration of TMAH is 5% (v/v which can immobilize up to 84.71% of GOX. X-ray diffraction (XRD and Fourier Transforms Infrared Spectroscopy (FTIR studies have been carried out to observe the structural changes in bentonite due to TMAH modification. The obtained immobilized GOX show the optimum catalytic activity on reaction temperature of 40-50 °C and pH of 7. The immobilized GOX kinetics at the optimum conditions determined the Km and Vmax value to be 4.96x10-2 mM and 4.99x10-3 mM.min-1 respectively. In addition, the immobilized GOX on TMAH-modified bentonite is stable enough so it could be re-used six times before its activity decreased by 39.44%.

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.

Extracellular polymeric substances from copper-tolerance Sinorhizobium meliloti immobilize Cu{sup 2+}

Energy Technology Data Exchange (ETDEWEB)

Hou, Wenjie; Ma, Zhanqiang; Sun, Liangliang; Han, Mengsha; Lu, Jianjun; Li, Zhenxiu; Mohamad, Osama Abdalla; Wei, Gehong, E-mail: weigehong@nwsuaf.edu.cn

2013-10-15

Highlights: • EPS produced by Sinorhizobium meliloti CCNWSX0020 restricts uptake of Cu{sup 2+}. • We focused on the EPS, which is divided into three main parts. • LB-EPS played a more important role than S-EPS and TB-EPS in Cu{sup 2+} immobilization. • Proteins and carbohydrates were the main extracellular compounds which had functional groups such as carboxyl (-COOH), hydroxyl (-OH), and amide (N-H), primarily involved in metal ion binding. -- Abstract: The copper tolerance gene of wild-type heavy metal-tolerance Sinorhizobium meliloti CCNWSX0020 was mutated by transposon Tn5-a. The mutant was sensitive up to 1.4 mM Cu{sup 2+}. Production, components, surface morphology, and functional groups of extracellular polymeric substances (EPS) of the wild-type strains were compared with sensitive mutant in immobilization of Cu{sup 2+}. EPS produced by S. meliloti CCNWSX0020 restricts uptake of Cu{sup 2+}. The cell wall EPS were categorized based on the compactness and fastness: soluble EPS (S-EPS), loosely bound EPS (LB-EPS), and tightly bound EPS (TB-EPS). LB-EPS played a more important role than S-EPS and TB-EPS in Cu{sup 2+} immobilization. Scanning electron microscopy (SEM) analysis LB-EPS had rough surface and many honeycomb pores, making them conducive to copper entry; therefore, they may play a role as a microbial protective barrier. Fourier transform-infrared (FT-IR) analysis further confirm that proteins and carbohydrates were the main extracellular compounds which had functional groups such as carboxyl (-COOH), hydroxyl (-OH), and amide (N-H), primarily involved in metal ion binding.

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.

A comparative study of the refractive index of silk protein thin films towards biomaterial based optical devices

Science.gov (United States)

Bucciarelli, A.; Mulloni, V.; Maniglio, D.; Pal, R. K.; Yadavalli, V. K.; Motta, A.; Quaranta, A.

2018-04-01

Over the last two decades, silk fibroin has been exploited as a versatile optical material in biological applications due to a combination of unique properties. Recently, protocols have been developed to produce a silk fibroin negative tone resist that is UV crosslinkable, thereby allowing micro and nanoscale patterning of the protein using traditional photolithographic tools. The same protocol has been applied to the silk protein sericin to develop a sericin resist. Despite the immense potential of these biomaterials to develop micro optical patterns on silicon and glass surfaces, as well as self-standing components, their refractive indexes are not well characterized. In this work, optimizing a method to obtain extremely smooth, thin films, the refractive index (RI) of fibroin and sericin proteins and resists were characterized using ellipsometry. The parameters of the Sellmeier and Cauchy dispersion laws have been determined to obtain the RI over a large wavelength range. A complete morphological study of the films has been conducted. In addition, the effect of solvent on the optical properties of silk fibroin and sericin thin films are reported, with differences in values explained by examining the change in the protein secondary structure.

Enhanced Biological Response of AVS-Functionalized Ti-6Al-4V Alloy through Covalent Immobilization of Collagen.

Science.gov (United States)

Rezvanian, Parsa; Daza, Rafael; López, Patricia A; Ramos, Milagros; González-Nieto, Daniel; Elices, Manuel; Guinea, Gustavo V; Pérez-Rigueiro, José

2018-02-20

This study presents the development of an efficient procedure for covalently immobilizing collagen molecules on AVS-functionalized Ti-6Al-4V samples, and the assessment of the survival and proliferation of cells cultured on these substrates. Activated Vapor Silanization (AVS) is a versatile functionalization technique that allows obtaining a high density of active amine groups on the surface. A procedure is presented to covalently bind collagen to the functional layer using EDC/NHS as cross-linker. The covalently bound collagen proteins are characterized by fluorescence microscopy and atomic force microscopy and their stability is tested. The effect of the cross-linker concentration on the process is assessed. The concentration of the cross-linker is optimized and a reliable cleaning protocol is developed for the removal of the excess of carbodiimide from the samples. The results demonstrate that the covalent immobilization of collagen type I on Ti-6Al-4V substrates, using the optimized protocol, increases the number of viable cells present on the material. Consequently, AVS in combination with the carbodiimide chemistry appears as a robust method for the immobilization of proteins and, for the first time, it is shown that it can be used to enhance the biological response to the material.

Raman optical activity of proteins and glycoproteins

International Nuclear Information System (INIS)

Smyth, E.

2000-03-01

Raman optical activity (ROA), measured in this project as a small difference in the intensity of Raman scattering from chiral molecules in right- and left-circularly polarised incident laser light, offers the potential to provide more information about the structure of biological molecules in aqueous solution than conventional spectroscopic techniques. Chapter one contains a general discussion of the relative merits of different spectroscopic techniques for structure determination of biomolecules, as well as a brief introduction to ROA. In Chapter two a theoretical analysis of ROA is developed, which extends the discussion in chapter one. The spectrometer setup and sample preparation is then discussed in chapter three. Instrument and sample conditions are monitored to ensure that the best results are obtained. As with any experimental project problems occur, which may result in a degradation of the spectra obtained. The cause of these problems was explored and remedied whenever possible. Chapter four introduces a brief account of protein, glycoprotein and carbohydrate structure and function, with a particular emphasis on the structure of proteins. In the remaining chapters experimental ROA results on proteins and glycoproteins, with some carbohydrate samples, from a wide range of sources are examined. For example, in chapter five some β-sheet proteins are examined. Structural features in these proteins are examined in the extended amide III region of their ROA spectra, revealing that ROA is sensitive to the rigidity or flexibility inherent in proteins. Chapter six concentrates on a group of proteins (usually glycoproteins) known as the serine proteinase inhibitors (serpins). Medically, the serpins are one of the most important groups of proteins of current interest, with wide-ranging implications in conditions such as Down's syndrome, Alzheimer's disease, and emphysema with associated cirrhosis of the liver. With favourable samples and conditions ROA may offer the

Immobilization thresholds of electrofishing relative to fish size

Science.gov (United States)

Dolan, C.R.; Miranda, L.E.

2003-01-01

Fish size and electrical waveforms have frequently been associated with variation in electrofishing effectiveness. Under controlled laboratory conditions, we measured the electrical power required by five electrical waveforms to immobilize eight fish species of diverse sizes and shapes. Fish size was indexed by total body length, surface area, volume, and weight; shape was indexed by the ratio of body length to body depth. Our objectives were to identify immobilization thresholds, elucidate the descriptors of fish size that were best associated with those immobilization thresholds, and determine whether the vulnerability of a species relative to other species remained constant across electrical treatments. The results confirmed that fish size is a key variable controlling the immobilization threshold and further suggested that the size descriptor best related to immobilization is fish volume. The peak power needed to immobilize fish decreased rapidly with increasing fish volume in small fish but decreased slowly for fish larger than 75-100 cm 3. Furthermore, when we controlled for size and shape, different waveforms did not favor particular species, possibly because of the overwhelming effect of body size. Many of the immobilization inconsistencies previously attributed to species might simply represent the effect of disparities in body size.

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.)

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.

Immobilization of Isolated Lipase From Moldy Copra (Aspergillus Oryzae

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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.

Magnetic particles-based biosensor for biogenic amines using an optical oxygen sensor as a transducer

International Nuclear Information System (INIS)

Pospiskova, K.; Sebela, M.; Safarik, I.; Kuncova, G.

2013-01-01

We have developed a fibre optic biosensor with incorporated magnetic microparticles for the determination of biogenic amines. The enzyme diamine oxidase from Pisum sativum was immobilized either on chitosan-coated magnetic microparticles or on commercial microbeads modified with a ferrofluid. Both the immobilized enzyme and the ruthenium complex were incorporated into a UV-cured inorganic-organic polymer composite and deposited on a lens that was connected, by optical fibres, to an electro-optical detector. The enzyme catalyzes the oxidation of amines under consumption of oxygen. The latter was determined by measuring the quenched fluorescence lifetime of the ruthenium complex. The limits of detection for the biogenic amines putrescine and cadaverine are 25-30 μmol L -1 , and responses are linear up to a concentration of 1 mmol L -1 . (author)

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....

Transition dipole coupling modeling of optical activity enhancements in macromolecular protein systems

Czech Academy of Sciences Publication Activity Database

Průša, Jiří; Bouř, Petr

2018-01-01

Roč. 30, č. 1 (2018), s. 55-64 ISSN 0899-0042 R&D Projects: GA ČR GA15-09072S; GA MŠk(CZ) LTC17012 Institutional support: RVO:61388963 Keywords : optical activity * vibrational circular dichroism * protein fibrils Subject RIV: BO - Biophysics OBOR OECD: Biophysics Impact factor: 1.956, year: 2016

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

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

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

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

Polymethacrylate Coated Electrospun PHB Fibers as a Functionalized Platform for Bio-Diagnostics: Confirmation Analysis on the Presence of Immobilized IgG Antibodies against Dengue Virus

Directory of Open Access Journals (Sweden)

Samira Hosseini

2017-10-01

Full Text Available In this article, a combination of far field electrospinning (FFES and free-radical polymerization has been used to create a unique platform for protein immobilization via the physical attachment of biomolecules to the surface of the fiber mats. The large specific surface area of the fibers with its tailored chemistry provides a desirable platform for effective analyte-surface interaction. The detailed analysis of protein immobilization on a newly developed bio-receptive surface plays a vital role to gauge its advantages in bio-diagnostic applications. We relied on scanning electron microscopy (SEM, diameter range analysis, and X-ray photoelectron spectroscopy (XPS, along with thermal gravimetric analysis (TGA, water-in-air contact angle analysis (WCA, Fourier transform infrared spectroscopy (FTIR, and atomic force microscopy (AFM to study our developed platforms and to provide valuable information regarding the presence of biomolecular entities on the surface. Detailed analyses of the fiber mats before and after antibody immobilization have shown obvious changes on the surface of the bioreceptive surface including: (i an additional peak corresponding to the presence of an antibody in TGA analysis; (ii extra FTIR peaks corresponding to the presence of antibodies on the coated fiber platforms; and (iii a clear alteration in surface roughness recorded by AFM analysis. Confirmation analyses on protein immobilization are of great importance as they underlay substantial grounds for various biosensing applications.

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 ...

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...

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

Metabolic alkalosis during immobilization in monkeys (M. nemestrina)

Science.gov (United States)

Young, D. R.; Yeh, I.; Swenson, R. S.

1983-01-01

The systemic and renal acid-base response of monkeys during ten weeks of immobilization was studied. By three weeks of immobilization, arterial pH and bicarbonate concentrations were elevated (chronic metabolic alkalosis). Net urinary acid excretion increased in immobilized animals. Urinary bicarbonate excretion decreased during the first three weeks of immobilization, and then returned to control levels. Sustained increases in urinary ammonium excretion were seen throughout the time duration of immobilization. Neither potassium depletion nor hypokalemia was observed. Most parameters returned promptly to the normal range during the first week of recovery. Factors tentatively associated with changes in acid-base status of monkeys include contraction of extracellular fluid volume, retention of bicarbonate, increased acid excretion, and possible participation of extrarenal buffers.

Immobilization of ammonia-oxidizing bacteria by polyvinyl alcohol and sodium alginate.

Science.gov (United States)

Dong, Yuwei; Zhang, Yanqiu; Tu, Baojun

Ammonia-oxidizing bacteria were immobilized by polyvinyl alcohol (PVA) and sodium alginate. The immobilization conditions and ammonia oxidation ability of the immobilized bacteria were investigated. The following immobilization conditions were observed to be optimal: PVA, 12%; sodium alginate, 1.1%; calcium chloride, 1.0%; inoculum concentration, 1.3 immobilized balls/mL of immobilized medium; pH, 10; and temperature, 30°C. The immobilized ammonia-oxidizing bacteria exhibited strong ammonia oxidation ability even after being recycled four times. The ammonia nitrogen removal rate of the immobilized ammonia-oxidizing bacteria reached 90.30% under the optimal immobilization conditions. When compared with ammonia-oxidizing bacteria immobilized by sodium alginate alone, the bacteria immobilized by PVA and sodium alginate were superior with respect to pH resistance, the number of reuses, material cost, heat resistance, and ammonia oxidation ability. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

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.

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

Fast axonal transport of 3H-leucin-labelled proteins in the unhurt and isolated optical nerve of rats

International Nuclear Information System (INIS)

Wagner, H.E.

1981-01-01

The distribution of radioactivity of amino acid molecules incorporated in protein after injection of 3 H-Leucin into the right bulb was investigated and determined along optical nerve after 1, 2, and 4 h. A slightly increased radioactivity at the point of entrance of the optical nerves into the optical duct was found. A slightly reduced axon diameter was discussed as a possible cause. The radioactivity brought into the optical nerve via the vascular system was determined by measuring the contralateral optical nerve. In relation to the axonally transported activity, it was low. The speed of the fast axonal transport is 168 mm/d. If the processes ruling the amino acids in the perikaryon are taken into consideration, the transport speed is 240 mm/d. The application of the protein synthesis prohibitor, Cycloheximide, 5 minutes after the injection of Leucinin completely prevented the appearance of axonally transported labelled proteins. When cycloheximide was administered 2 h after Leucin, a significantly loner radioactivity than in the nerve could be determined after another 2 h; i.e. the incorporation of Leucin was not completed yet after 2 h. The profile of active compounds was the same as in the control group. In other experiments, the axonal transport of labelled proteins in isolated optical nerve fibres was tested. If the separation was carried out 2 h after the injection of Leucin an extreme reduction in activity could be determined after 1 or 2 h. The continued distribution of activity after cycloheximide treatment and removal of perikarya in comparison with the control indicate the continuation of the transport, also after separation of the axon from the perikaryon. This means that, during the time of the experiment, the mechanism of the fast axonal transport functions independently of the perikaryon. (orig./MG) [de

Thermal stability of the immobilized fructosyltransferase from Rhodotorula sp

Directory of Open Access Journals (Sweden)

E Aguiar-Oliveira

2011-09-01

Full Text Available The thermal stability of the extracellular fructosyltransferase (FTase from Rhodotorula sp., recovered from cultivation medium by ethanol precipitation and immobilized onto niobium ore, was studied by Arrhenius plot, half - life profile, half - inactivation temperature (T50 and thermodynamic parameters. The Arrhenius plot showed two different behaviors with different deactivation energies (Ead only after immobilization, the transition occurring in the temperature interval between 51 and 52ºC. T50 for the free enzyme was estimated to be around 62ºC and, after immobilization, 66ºC. After 15 minutes at 52ºC, it was also possible to observe enzymatic activation for both the free and immobilized forms, but greater activation was achieved at pH 4.5 with the immobilized enzyme. Between 47 - 51ºC the immobilized enzyme was more stable than the free enzyme, with pH 6.0 being the more stable condition for the immobilized enzyme. However, above 52ºC the free form was more stable.

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....

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

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.

An automated wide-field time-gated optically sectioning fluorescence lifetime imaging multiwell plate reader for high-content analysis of protein-protein interactions

Science.gov (United States)

Alibhai, Dominic; Kumar, Sunil; Kelly, Douglas; Warren, Sean; Alexandrov, Yuriy; Munro, Ian; McGinty, James; Talbot, Clifford; Murray, Edward J.; Stuhmeier, Frank; Neil, Mark A. A.; Dunsby, Chris; French, Paul M. W.

2011-03-01

We describe an optically-sectioned FLIM multiwell plate reader that combines Nipkow microscopy with wide-field time-gated FLIM, and its application to high content analysis of FRET. The system acquires sectioned FLIM images in fluorescent protein. It has been applied to study the formation of immature HIV virus like particles (VLPs) in live cells by monitoring Gag-Gag protein interactions using FLIM FRET of HIV-1 Gag transfected with CFP or YFP. VLP formation results in FRET between closely packed Gag proteins, as confirmed by our FLIM analysis that includes automatic image segmentation.

Unilateral hindlimb casting induced a delayed generalized muscle atrophy during rehabilitation that is prevented by a whey or a high protein diet but not a free leucine-enriched diet.

Directory of Open Access Journals (Sweden)

Hugues Magne

Full Text Available Sarcopenia is the general muscle mass and strength loss associated with ageing. Muscle atrophy could be made worse by exposure to acute periods of immobilization, because muscle disuse by itself is a stimulus for atrophy. Using a model of unilateral hindlimb casting in old adult rats, we have already demonstrated that the primary effect of immobilization was atrophy in the casted leg, but was also surprisingly associated with a retarded atrophy in the non-casted leg during rehabilitation. In search of mechanisms involved in this generalized atrophy, we demonstrated in the present study that contrary to pair-fed non-immobilized control animals, muscle protein synthesis in the non-immobilized limb was unable to adapt and to respond positively to food intake. Because pair-fed control rats did not lose muscle mass, this defect in muscle protein synthesis may represent one of the explanation for the muscle mass loss observed in the non-immobilized rats. Nevertheless, in order to stimulate protein turn over and generate a positive nitrogen balance required to maintain the whole muscle mass in immobilized rats, we tested a dietary free leucine supplementation (an amino acid known for its stimulatory effect on protein metabolism during the rehabilitation period. Leucine supplementation was able to overcome the anabolic resistance in the non-immobilized limb. A greater muscle protein synthesis up-regulation associated with a stimulation of the mTOR signalling pathway was indeed recorded but it remained inefficient to prevent the loss of muscle in the non-immobilized limb. By contrast, we demonstrated here that whey protein or high protein diets were able to prevent the muscle mass loss of the non-immobilized limb by sustaining muscle protein synthesis during the entire rehabilitation period.

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...

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…

Synthesis of an endothelial cell mimicking surface containing thrombomodulin and endothelial protein C receptor

Science.gov (United States)

Kador, Karl Erich

Synthetic materials for use in blood contacting applications have been studied for many years with limited success. One of the main areas of need for these materials is the design of synthetic vascular grafts for use in the hundreds of thousands of patients who have coronary artery bypass grafting, many without suitable veins for autologous grafts. The design of these grafts is constrained by two common modes of failure, the formation of intimal hyperplasia (IH) and thrombosis. IH formation has been previously linked to a mismatching of the mechanical properties of the graft and has been overcome by creating grafts using materials whose compliance mimics that of the native artery. Several techniques and surface modification have been designed to limit thrombosis on the surface of synthetic materials. One which has shown the greatest promise is the immobilization of Thrombomodulin (TM), a protein found on the endothelial cell membrane lining native blood vessels involved in the activation of the anticoagulant Protein C (PC). While TM immobilization has been shown to arrest thrombin formation and limit fibrous formations in in-vitro and in-vivo experiments, it has shown to be transport limiting under arterial flow. On the endothelial cell surface, TM is co-localized with Endothelial Protein C Receptor (EPCR), which increases PC transport onto the cell surface and increases PC activation via TM between 20-100 fold. This dissertation will describe the chemical modification of medical grade polyurethane (PU), whose compliance has been shown to match that of native arteries. This modification will enable the immobilization of two proteins on an enzymatically relevant scale estimated at less than 10 nm. This dissertation will further describe the immobilization of the proteins TM and EPCR, and analyze the ability of a surface co-immobilized with these proteins to activate the anticoagulant PC. Finally, it will compare the ability of this co-immobilized surface to delay

Covalent microcontact printing of proteins fro cell patterning

NARCIS (Netherlands)

Rozkiewicz, D.I.; Kraan, Yvonne M.; Werten, Marc W.T.; de Wolf, Frits A.; Subramaniam, Vinod; Ravoo, B.J.; Reinhoudt, David

2006-01-01

We describe a straightforward approach to the covalent immobilization of cytophilic proteins by microcontact printing, which can be used to pattern cells on substrates. Cytophilic proteins are printed in micropatterns on reactive self-assembled monolayers by using imine chemistry. An

Immobilization of calcium and phosphate ions improves the osteoconductivity of titanium implants

International Nuclear Information System (INIS)

Sunarso; Toita, Riki; Tsuru, Kanji; Ishikawa, Kunio

2016-01-01

In this work, to elevate weak osteoconductivity of titanium (Ti) implant, we prepared a Ti implant having both calcium and phosphate ions on its surface. To modify calcium and phosphate ions onto Ti, phosphate ions were first immobilized by treating the Ti with a NaH 2 PO 4 solution, followed by CaCl 2 treatment to immobilize calcium ions, which created the calcium and phosphate ions-modified Ti (Ca-P-Ti). X-ray photoelectron spectroscopy and thin-layer X-ray diffraction measurement confirmed that both phosphate and calcium ions were co-immobilized onto the Ti surface on the molecular level. Three-hour after seeding MC3T3-E1 murine pre-osteoblast cells on substrates, cell number on Ca-P-Ti was much larger than that of Ti and phosphate-modified Ti (P-Ti), but was similar to that of calcium-modified Ti (Ca-Ti). Also, MC3T3-E1 cells on Ca-P-Ti expressed larger amount of vinculin, a focal adhesion protein, than those on other substrates, probably resulting in larger cell size as well as greater cell proliferation on Ca-P-Ti than those on other substrates. Alkaline phosphatase activity of cells on Ca-P-Ti was greater than those on Ti and P-Ti, but was almost comparable to that of Ca-Ti. Moreover, the largest amount of bone-like nodule formation was observed on Ca-P-Ti. These results provide evidence that calcium and phosphate ions-co-immobilization onto Ti increased the osteoconductivity of Ti by stimulating the responses of pre-osteoblast cells. This simple modification would be promising technique for bone tissue implant including dental and orthopedic implants. - Highlights: • Phosphate and calcium ions have been successfully co-immobilize on Ti surface. • Co-immobilization of Ca and phosphate ions (Ca-P-Ti) did not alter the original surface morphology. • Ca-P-Ti significantly improved initial MC3T3-E1 cell adhesion. • Ca-P-Ti demonstrated remarkable cell proliferation, differentiation and mineralization. • Overall, Ca-P-Ti would be a promising bone

Novel regenerative large-volume immobilized enzyme reactor: preparation, characterization and application.

Science.gov (United States)

Ruan, Guihua; Wei, Meiping; Chen, Zhengyi; Su, Rihui; Du, Fuyou; Zheng, Yanjie

2014-09-15

A novel large-volume immobilized enzyme reactor (IMER) on small column was prepared with organic-inorganic hybrid silica particles and applied for fast (10 min) and oriented digestion of protein. At first, a thin enzyme support layer was formed in the bottom of the small column by polymerization with α-methacrylic acid and dimethacrylate. After that, amino SiO2 particles was prepared by the sol-gel method with tetraethoxysilane and 3-aminopropyltriethoxysilane. Subsequently, the amino SiO2 particles were activated by glutaraldehyde for covalent immobilization of trypsin. Digestive capability of large-volume IMER for proteins was investigated by using bovine serum albumin (BSA), cytochrome c (Cyt-c) as model proteins. Results showed that although the sequence coverage of the BSA (20%) and Cyt-c (19%) was low, the large-volume IMER could produce peptides with stable specific sequence at 101-105, 156-160, 205-209, 212-218, 229-232, 257-263 and 473-451 of the amino sequence of BSA when digesting 1mg/mL BSA. Eight of common peptides were observed during each of the ten runs of large-volume IMER. Besides, the IMER could be easily regenerated by reactivating with GA and cross-linking with trypsin after breaking the -C=N- bond by 0.01 M HCl. The sequence coverage of BSA from regenerated IMER increased to 25% comparing the non-regenerated IMER (17%). 14 common peptides. accounting for 87.5% of first use of IMER, were produced both with IMER and regenerated IMER. When the IMER was applied for ginkgo albumin digestion, the sequence coverage of two main proteins of ginkgo, ginnacin and legumin, was 56% and 55%, respectively. (Reviewer 2) Above all, the fast and selective digestion property of the large-volume IMER indicated that the regenerative IMER could be tentatively used for the production of potential bioactive peptides and the study of oriented protein digestion. Copyright © 2014 Elsevier B.V. All rights reserved.

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 ...