140 results on '"Channing R. Robertson"'
Search Results
2. Organic Thin Films
- Author
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CURTIS W. FRANK, J. D. Swalen, Werner A. Goedel, J. P. Kampf, T. L. Einloth, Curtis W. Frank, Yufei Li, Jae-Min Oh, J. A. Moore, Kang Sub Yim, Carlton F. Brooks, Gerald G. Fuller, Curtis W. Frank, Channing R. Robertson, Noemi Zenou, Alexander Zelichenok, Shlomo Yitzchaik, Rami Cohen, David Cahen, Je and CURTIS W. FRANK, J. D. Swalen, Werner A. Goedel, J. P. Kampf, T. L. Einloth, Curtis W. Frank, Yufei Li, Jae-Min Oh, J. A. Moore, Kang Sub Yim, Carlton F. Brooks, Gerald G. Fuller, Curtis W. Frank, Channing R. Robertson, Noemi Zenou, Alexander Zelichenok, Shlomo Yitzchaik, Rami Cohen, David Cahen, Je
- Published
- 1998
3. Engineering of a miniaturized, robotic clinical laboratory
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Pradeep L. Ramachandran, Elizabeth A. Holmes, Chinmay Pangarkar, Karthik Jayasurya, Jared O'Leary, Chandan Shee, Alphonso Nguyen, Lucie S. Lee, Laura Hyland, Marilyn B. Nourse, Kevin D. Ha, Steven Chow, Samartha Anekal, Pradeep Bhatta, Steven F. Gessert, Ran Hu, Kate Engel, Renuka Shenoy, Yang Lily Liu, Bernardo Sosa-Padilla, Ushati Das, Amy Yuan, Jerald F. Sapida, Joy Roy, Peter Zhao, Lorraine Tran, Dariusz Wodziak, Amanda Trent, Daniel L. Young, Shekar Chandrasekaran, Amy R. Rappaport, Ken Quon, Andrew N. Kim, Yutao Chen, Timothy Michael Kemp, Arvind Jammalamadaka, Thomas C. Waggoner, Jocelyn A. Bailey, Channing R. Robertson, Xinwei Gong, Erez Galil, Nikolay V. Sergeev, Devayani Bhave, Sharada Sivaraman, and Paul Patel
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0301 basic medicine ,Analyte ,Spectrum analyzer ,business.industry ,Computer science ,Small footprint ,Biomedical Engineering ,Pharmaceutical Science ,030204 cardiovascular system & hematology ,Molecular diagnostics ,Laboratory testing ,Automation ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Virus type ,Minilab ,business ,Computer hardware ,Biotechnology - Abstract
The ability to perform laboratory testing near the patient and with smaller blood volumes would benefit patients and physicians alike. We describe our design of a miniaturized clinical laboratory system with three components: a hardware platform (ie, the miniLab) that performs preanalytical and analytical processing steps using miniaturized sample manipulation and detection modules, an assay-configurable cartridge that provides consumable materials and assay reagents, and a server that communicates bidirectionally with the miniLab to manage assay-specific protocols and analyze, store, and report results (i.e., the virtual analyzer). The miniLab can detect analytes in blood using multiple methods, including molecular diagnostics, immunoassays, clinical chemistry, and hematology. Analytical performance results show that our qualitative Zika virus assay has a limit of detection of 55 genomic copies/ml. For our anti-herpes simplex virus type 2 immunoglobulin G, lipid panel, and lymphocyte subset panel assays, the miniLab has low imprecision, and method comparison results agree well with those from the United States Food and Drug Administration-cleared devices. With its small footprint and versatility, the miniLab has the potential to provide testing of a range of analytes in decentralized locations.
- Published
- 2018
4. Biological Applications of Total Internal Reflection Fluorescence
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Channing R. Robertson and Seth A. Darst
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Optics ,Total internal reflection fluorescence microscope ,Materials science ,business.industry ,business - Published
- 2018
5. Engineering of a miniaturized, robotic clinical laboratory
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Marilyn B, Nourse, Kate, Engel, Samartha G, Anekal, Jocelyn A, Bailey, Pradeep, Bhatta, Devayani P, Bhave, Shekar, Chandrasekaran, Yutao, Chen, Steven, Chow, Ushati, Das, Erez, Galil, Xinwei, Gong, Steven F, Gessert, Kevin D, Ha, Ran, Hu, Laura, Hyland, Arvind, Jammalamadaka, Karthik, Jayasurya, Timothy M, Kemp, Andrew N, Kim, Lucie S, Lee, Yang Lily, Liu, Alphonso, Nguyen, Jared, O'Leary, Chinmay H, Pangarkar, Paul J, Patel, Ken, Quon, Pradeep L, Ramachandran, Amy R, Rappaport, Joy, Roy, Jerald F, Sapida, Nikolay V, Sergeev, Chandan, Shee, Renuka, Shenoy, Sharada, Sivaraman, Bernardo, Sosa-Padilla, Lorraine, Tran, Amanda, Trent, Thomas C, Waggoner, Dariusz, Wodziak, Amy, Yuan, Peter, Zhao, Daniel L, Young, Channing R, Robertson, and Elizabeth A, Holmes
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Research Report ,molecular diagnostics ,hematology ,laboratory testing ,diagnostics ,Research Reports ,immunoassay ,clinical chemistry ,automation - Abstract
The ability to perform laboratory testing near the patient and with smaller blood volumes would benefit patients and physicians alike. We describe our design of a miniaturized clinical laboratory system with three components: a hardware platform (ie, the miniLab) that performs preanalytical and analytical processing steps using miniaturized sample manipulation and detection modules, an assay‐configurable cartridge that provides consumable materials and assay reagents, and a server that communicates bidirectionally with the miniLab to manage assay‐specific protocols and analyze, store, and report results (i.e., the virtual analyzer). The miniLab can detect analytes in blood using multiple methods, including molecular diagnostics, immunoassays, clinical chemistry, and hematology. Analytical performance results show that our qualitative Zika virus assay has a limit of detection of 55 genomic copies/ml. For our anti‐herpes simplex virus type 2 immunoglobulin G, lipid panel, and lymphocyte subset panel assays, the miniLab has low imprecision, and method comparison results agree well with those from the United States Food and Drug Administration‐cleared devices. With its small footprint and versatility, the miniLab has the potential to provide testing of a range of analytes in decentralized locations.
- Published
- 2017
6. The Role of Electrostatic Interactions in Protease Surface Diffusion and the Consequence for Interfacial Biocatalysis
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Luis G. Cascao-Pereira, Bob E. Feller, Curtis W. Frank, James T. Kellis, and Channing R. Robertson
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Surface Properties ,Diffusion ,Static Electricity ,Analytical chemistry ,Reaction rate ,Adsorption ,Electrochemistry ,Animals ,General Materials Science ,Surface plasmon resonance ,Spectroscopy ,Cellulomonas ,Surface diffusion ,Chemistry ,Osmolar Concentration ,Substrate (chemistry) ,Surfaces and Interfaces ,Condensed Matter Physics ,Electrostatics ,Kinetics ,Chemical engineering ,Ionic strength ,Biocatalysis ,Cattle ,Peptide Hydrolases - Abstract
This study examines the influence of electrostatic interactions on enzyme surface diffusion and the contribution of diffusion to interfacial biocatalysis. Surface diffusion, adsorption, and reaction were investigated on an immobilized bovine serum albumin (BSA) multilayer substrate over a range of solution ionic strength values. Interfacial charge of the enzyme and substrate surface was maintained by performing the measurements at a fixed pH; therefore, electrostatic interactions were manipulated by changing the ionic strength. The interfacial processes were investigated using a combination of techniques: fluorescence recovery after photobleaching, surface plasmon resonance, and surface plasmon fluorescence spectroscopy. We used an enzyme charge ladder with a net charge ranging from -2 to +4 with respect to the parent to systematically probe the contribution of electrostatics in interfacial enzyme biocatalysis on a charged substrate. The correlation between reaction rate and adsorption was determined for each charge variant within the ladder, each of which displayed a maximum rate at an intermediate surface concentration. Both the maximum reaction rate and adsorption value at which this maximum rate occurs increased in magnitude for the more positive variants. In addition, the specific enzyme activity increased as the level of adsorption decreased, and for the lowest adsorption values, the specific enzyme activity was enhanced compared to the trend at higher surface concentrations. At a fixed level of adsorption, the specific enzyme activity increased with positive enzyme charge; however, this effect offers diminishing returns as the enzyme becomes more highly charged. We examined the effect of electrostatic interactions on surface diffusion. As the binding affinity was reduced by increasing the solution ionic strength, thus weakening electrostatic interaction, the rate of surface diffusion increased considerably. The enhancement in specific activity achieved at the lowest adsorption values is explained by the substantial rise in surface diffusion at high ionic strength due to decreased interactions with the surface. Overall, knowledge of the electrostatic interactions can be used to control surface parameters such as surface concentration and surface diffusion, which intimately correlate with surface biocatalysis. We propose that the maximum reaction rate results from a balance between adsorption and surface diffusion. The above finding suggests enzyme engineering and process design strategies for improving interfacial biocatalysis in industrial, pharmaceutical, and food applications.
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- 2010
7. Open Doorway to Truth: Legacy of the Minnesota Tobacco Trial
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Richard D. Hurt, Jon O. Ebbert, Monique E. Muggli, Nikki J. Lockhart, and Channing R. Robertson
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General Medicine - Published
- 2009
8. Guest Editorial Special Section on Drug Delivery Automation
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T. C. Yih, Mingjun Zhang, Channing R. Robertson, Chiming Wei, Liwei Lin, and Deirdre R. Meldrum
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Engineering management ,Engineering ,Control and Systems Engineering ,Home automation ,business.industry ,Section (typography) ,Drug delivery ,Special section ,Mechanical engineering ,Electrical and Electronic Engineering ,business ,Theme (computing) ,Automation - Abstract
The central theme in this special section is recent progress in automation for drug delivery. There are five papers in this section.
- Published
- 2009
9. Enzymatic Proteolysis of a Surface-Bound α-Helical Polypeptide
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Curtis W. Frank, James T. Kellis, Luis G. Cascao-Pereira, Channing R. Robertson, and Jasper O. Hardesty
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Models, Molecular ,Circular dichroism ,Proteases ,Proteolysis ,medicine.medical_treatment ,Analytical chemistry ,Bacillus ,Protein Structure, Secondary ,Protein structure ,Catalytic Domain ,Electrochemistry ,medicine ,General Materials Science ,Protein Structure, Quaternary ,Protein secondary structure ,Spectroscopy ,Protease ,medicine.diagnostic_test ,Chemistry ,Circular Dichroism ,Hydrolysis ,Serine Endopeptidases ,Substrate (chemistry) ,Surfaces and Interfaces ,Condensed Matter Physics ,Ionic strength ,Biophysics ,Peptides - Abstract
In this work, we studied the interactions of enzymes with model substrate surfaces using label-free techniques. Our model system was based on serine proteases (a class of enzymes that digests proteins) and surface-bound polypeptide substrates. While previous studies have focused on bulk media factors such as pH, ionic strength, and surfactants, this study focuses on the role of the surface-bound substrate itself. In particular, we assess how the substrate density of a polypeptide with an alpha-helical secondary structure influences surface reactivity. An alpha-helical secondary structure was chosen based on literature indicating that stable alpha-helices can resist enzymatic digestion. To investigate the protease resistance of a surface-bound a-helix, we designed an a-helical polypeptide (SS-polypeptide, where SS = disulfide), used it to form films of varying surface coverage and then measured responses of the films to enzymatic exposure. Using quartz-crystal microbalance with dissipation (QCM-D), angle-resolved X-ray photoelectron spectroscopy (AR-XPS), grazing-angle infrared spectroscopy (GAIRS), and other techniques, we characterized the degradation of films to determine how the lateral packing density of the surface-bound SS-polypeptide substrate affected surface proteolysis. Characterization of pure SS-polypeptide films indicated dense packing of helices that maintained their helical structure and were generally oriented normal to the surface. We found that films of pure SS-polypeptide significantly resisted enzymatic digestion, while incorporation of very minor amounts of a diluent in such films resulted in rapid digestion. In part, this may be due to the need for the enzyme to bind several peptides along the peptide substrate within the cleft for digestion to occur. Only SS-polypeptide films that were densely packed and did not permit catalytic access to multiple peptides (e.g., terminal peptides only) were resistant to enzymatic proteolysis.
- Published
- 2008
10. Fluorescence Quantification for Surface Plasmon Excitation
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Wolfgang Knoll, Luis G. Cascao-Pereira, Bob E. Feller, James T. Kellis, Channing R. Robertson, and Curtis W. Frank
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Chemistry ,Surface plasmon ,Analytical chemistry ,Resonance ,Surfaces and Interfaces ,Condensed Matter Physics ,Fluorescence ,Fluorescence spectroscopy ,Adsorption ,Electrochemistry ,General Materials Science ,Fluorescence cross-correlation spectroscopy ,Surface plasmon resonance ,Spectroscopy ,Localized surface plasmon - Abstract
Surface plasmon resonance and surface plasmon fluorescence spectroscopy in combination have the potential to distinguish multicomponent surface processes. However, surface intensity variations from resonance angle shifts lead to a nonlinear response in the fluorescence intensity. We report a method to account for surface intensity variations using the experimentally measured relationship between fluorescence and reflectivity. We apply this method to monitor protease adsorption and proteolytic substrate degradation simultaneously. Multilayer protein substrates are prepared for these degradation studies using a layer-by-layer technique.
- Published
- 2008
11. Downloadable computer models for renal replacement therapy
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Timothy W. Meyer, Jason Walther, Channing R. Robertson, D.W. Bartlett, W. Chew, and Thomas H. Hostetter
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medicine.medical_specialty ,Time Factors ,Metabolic Clearance Rate ,medicine.medical_treatment ,Diffusion ,030232 urology & nephrology ,Ultrafiltration ,Thermodynamics ,Dialysate flow ,030204 cardiovascular system & hematology ,Artificial kidney ,Renal Circulation ,hemofiltration ,03 medical and health sciences ,0302 clinical medicine ,Renal Dialysis ,Mass transfer ,medicine ,Animals ,Humans ,Computer Simulation ,Renal replacement therapy ,computer ,Mathematics ,hemodialysis ,Mathematical model ,modeling ,Membranes, Artificial ,Models, Theoretical ,Hemodialysis Solutions ,Surgery ,Renal Replacement Therapy ,Hematocrit ,Nephrology ,Software ,Production rate ,Glomerular Filtration Rate - Abstract
Mathematical models can predict solute clearances and solute concentrations during renal replacement therapy. At present, however, most nephrologists cannot use these models because they require mathematical software. In this report, we describe models of solute transport by convection and diffusion adapted to run on the commonly available software program Excel® for Macintosh® computers and PCs running Windows®. Two programs have been created that can be downloaded from http://www.stanford.edu/~twmeyer/ or http://dev.satellitehealth.com/research/journal.asp. The first, called ‘Dr Addis Clearance Calculator', calculates clearance values from inputs including the blood flow Q b , the hematocrit, the ultrafiltration rate Q f , the dialysate flow rate Q d , the reflection coefficient σ and the mass transfer area coefficient K o A for the solute of interest, and the free fraction f if the solute is protein bound. Solute concentration profiles along the length of the artificial kidney are displayed graphically. The second program, called ‘Dr Coplon Dialysis Simulator', calculates plasma solute concentrations from the clearance values obtained by the first program and from additional input values including the number of treatments per week, the duration of the treatments, and the solute's production rate and volumes of distribution. The program calculates the time-averaged solute concentration and provides a graphic display of the solute concentration profile through a week-long interval.
- Published
- 2006
- Full Text
- View/download PDF
12. Simultaneous Observation of Enzyme Surface Diffusion and Surface Reaction Using Microfluidic Patterning of Substrate Surfaces
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James T. Kellis, Alice P. Gast, A. J. Poulose, Jerry M. Thomas, Elizabeth A. Holmes, Shaunak Roy, and Channing R. Robertson
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Surface diffusion ,biology ,Surface Properties ,Chemistry ,Diffusion ,Microfluidics ,Subtilisin ,Analytical chemistry ,Substrate (chemistry) ,Serum Albumin, Bovine ,Microfluidic Analytical Techniques ,Enzymes ,Analytical Chemistry ,Reaction rate ,Adsorption ,Chemical engineering ,biology.protein ,Animals ,Cattle ,Reactivity (chemistry) ,Bovine serum albumin ,Fluorescence Recovery After Photobleaching - Abstract
We present a study of the simultaneous observation of protease reaction and surface diffusion as the enzyme interacts with a model substrate surface. We use micro-fluidic patterning to decorate a bovine serum albumin substrate surface with stripes of adsorbed enzyme in the absence of physical barriers. Spreading of the enzyme from the initial striped region indicates surface diffusion, while removal of the substrate provides a measure of reactivity. Microfluidic patterning provides a means to determine the relative importance of enzyme adsorption, surface diffusion, and reaction on the rate of substrate removal.
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- 2005
13. Helical crystallization on lipid nanotubes: Streptavidin as a model protein
- Author
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Sammy J. Farah, Thanh X. Dang, Bridget Carragher, Alice P. Gast, Edward H. Egelman, Elizabeth M. Wilson-Kubalek, and Channing R. Robertson
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Streptavidin ,Materials science ,Protein Array Analysis ,Quantitative Biology::Cell Behavior ,law.invention ,Quantitative Biology::Subcellular Processes ,chemistry.chemical_compound ,Protein structure ,Structural Biology ,law ,Monolayer ,Nanotechnology ,Molecule ,Crystallization ,Quantitative Biology::Biomolecules ,Nanotubes ,Intermolecular force ,Lipids ,Condensed Matter::Soft Condensed Matter ,Crystallography ,chemistry ,Biotinylation ,Mutation ,Protein microarray - Abstract
In this study, we use streptavidin (SA) as a model system to study helical protein array formation on lipid nanotubes, an alternative to 2D studies on lipid monolayers. We demonstrate that wild-type and a mutant form of SA form helical arrays on biotinylated lipid nanotubes. 3D maps from helical arrays of wild-type and mutant SA were reconstructed using two different approaches: Fourier-Bessel methods and an iterative single particle algorithm. The maps show that wild-type and mutant streptavidin molecules order differently. The molecular packing arrangements of SA on the surface of the lipid nanotubes differ from previously reported lattice packing of SA on biotinylated monolayers. Helical crystallization on lipid nanotubes presents an alternative platform to explore fundamentals of protein ordering, intermolecular protein interaction and phase behavior. We demonstrate that lipid nanotubes offer a robust and reproducible substrate for forming helical protein arrays which present a means for studying protein structure and structure-function relationships.
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- 2005
14. Increasing Dialysate Flow and Dialyzer Mass Transfer Area Coefficient to Increase the Clearance of Protein-bound Solutes
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Thomas H. Hostetter, Timothy W. Meyer, Evonne C Leeper, Yiming Z. Lit, Channing R. Robertson, Thomas A. Depner, and Derek W Bartlett
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medicine.medical_specialty ,Time Factors ,Phenolsulfonphthalein ,chemistry.chemical_compound ,Renal Dialysis ,Albumins ,Dialysis Solutions ,Mass transfer ,medicine ,Humans ,Urea ,Mass transfer coefficient ,Phenol red ,Creatinine ,Models, Statistical ,Chromatography ,Extraction (chemistry) ,Albumin ,Proteins ,Membranes, Artificial ,General Medicine ,Models, Theoretical ,Blood proteins ,Surgery ,chemistry ,Nephrology ,Kidney Failure, Chronic ,Hemofiltration ,Protein Binding - Abstract
Clinical hemodialysis systems achieve high single pass extraction of small solutes that are not bound to plasma proteins. But they clear protein-bound solutes much less effectively. This study examines the extent to which clearance of a protein-bound test solute is improved by increasing the dialyzer mass transfer area coefficient (KoA) and the dialysate flow rate (Qd). A reservoir containing test solutes and artificial plasma with albumin concentration approximately 4 g/dl was dialyzed with a standard clinical dialysate delivery system. The clearance of phenol red (ClPR) was compared with the clearances of urea and creatinine at a plasma flow rate (Qp) of 200 ml/min with varying values of KoA and Qd. ClPR increased from 11 +/- 2 ml/min to 23 +/- 2 ml/min when KoA for phenol red, KoAPR, was increased from 238 to 640 ml/min and Qd was increased from 286 +/- 6 ml/min to 734 +/- 9 ml/min. Increasing either KoAPR or Qd alone had lesser effects. Clearance values for phenol red were much lower than clearance values for the unbound solutes urea and creatinine, which ranged from 150 to 200 ml/min and were less affected by varying KoA and Qd. A mathematical model was developed to predict ClPR from values of Qp, Qd, the fraction of phenol red bound to albumin (94% +/- 1%) and KoAPR. The model accurately predicts the pattern of measured results and shows further that ClPR can be made to approach Qp only by very large increases in both KoAPR and Qd.
- Published
- 2004
15. Surface Plasmon Resonance/Surface Plasmon Enhanced Fluorescence: An Optical Technique for the Detection of Multicomponent Macromolecular Adsorption at the Solid/Liquid Interface
- Author
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Joon-H. Kim,†,‡, and Channing R. Robertson, A. J. Poulose, Alice P. Gast, James T. Kellis, and Shaunak Roy
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biology ,Chemistry ,education ,Surface plasmon ,Analytical chemistry ,Substrate (chemistry) ,Surfaces and Interfaces ,Condensed Matter Physics ,Fluorescence ,Adsorption ,Electrochemistry ,biology.protein ,General Materials Science ,Surface plasmon resonance ,Spectroscopy ,Avidin ,Macromolecule ,Localized surface plasmon - Abstract
We describe an optical technique for the measurement of macromolecular adsorption at the solid/liquid interface when multiple species are present. The technique combines surface plasmon resonance (SPR) with simultaneous surface plasmon enhanced fluorescence (SPEF). The relative ease of construction and linear correlation between SPR and SPEF signals make the technique amenable for coadsorption studies or multiple ligand binding experiments. Here, we demonstrate the utility of the technique with a biotin/avidin/BSA “sandwich” experiment. We then apply SPR/SPEF for the simultaneous monitoring of enzyme adsorption and substrate cleavage of a protease interacting with a substrate surface.
- Published
- 2002
16. Protease Adsorption and Reaction on an Immobilized Substrate Surface
- Author
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Shaunak Roy, Joon-H. Kim,†,‡, A. J. Poulose, Channing R. Robertson, Alice P. Gast, and James T. Kellis
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biology ,Chemistry ,Subtilisin ,Substrate (chemistry) ,Surfaces and Interfaces ,Condensed Matter Physics ,Photochemistry ,Contact lens ,Adsorption ,Electrochemistry ,biology.protein ,Organic chemistry ,General Materials Science ,Enzyme kinetics ,Surface plasmon resonance ,Bovine serum albumin ,Spectroscopy ,Protein adsorption - Abstract
Enzymatic reactions with surface-bound substrates present an interesting problem in biomolecular surface science, as they require us to consider traditional enzyme kinetics in the context of protein adsorption. These reactions are important in such applications as detergent enzyme additives, food processing, and contact lens cleaning. We study the interaction of a serine protease (subtilisin) with an immobilized substrate (bovine serum albumin) surface through the simultaneous use of surface plasmon resonance and surface plasmon enhanced fluorescence techniques. We measure adsorbed enzyme concentrations and substrate cleavage rates in situ and compare the reactivities with those in solution. By varying the ionic strength of the reaction environment and studying several single point mutations of subtilisin, we find the adsorption behavior of the enzyme is strongly influenced by its electrostatic interactions with the charged bovine serum albumin surface. The surface reactivity of each of the mutants is cou...
- Published
- 2002
17. Surface Shear Rheology of a Polymerizable Lipopolymer Monolayer
- Author
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Gerald G. Fuller, Curtis W. Frank, Wolfgang Knoll, Jochen Thiele, Carlton F. Brooks, David F. O'Brien, and Channing R. Robertson
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chemistry.chemical_classification ,Materials science ,Rheometer ,Surfaces and Interfaces ,Polymer ,Dynamic mechanical analysis ,Condensed Matter Physics ,Shear modulus ,Light intensity ,chemistry ,Polymerization ,Monolayer ,Electrochemistry ,Side chain ,Organic chemistry ,General Materials Science ,Composite material ,Spectroscopy - Abstract
We have used an interfacial stress rheometer to study the changes in the mechanical shear properties of a Langmuir monolayer of a linear polymer with reactive side chains. The polymer investigated had a linear hydrophilicbackbone consisting of poly(ethylene imine-co-ethyl oxazoline) and contained 20% hydrophobic sorbyl side chains, making it amphiphilic and capable of forming stable monolayers at the air-water interface. The sorbyl moiety in the side chain was photopolymerizable at 254 nm. Upon exposure of the film to UV light, the surface dynamic shear modulus, G' s , which is a measure of the elasticity, increased 1000-fold to ∼2 mN/m. This large increase in elasticity arises from the formation of a network by reaction of side chains on different polymer backbones. Rubber-like properties were deduced from the frequency dependence of the moduli and from creep experiments, where elastic recoil could be observed upon removing the applied stress. Such a network could be formed over a wide range of surface pressures ranging from 5 to 25 mN/m. We investigated the cross-linking kinetics upon UV exposure as a function of temperature, initial surface pressure, and light intensity. We have developed a lumped kinetic model that agrees with the transient behavior of the storage modulus, our measure of the density of cross-link points. The reaction was independent of temperature (over 10-30 °C) and exhibited a first-order dependence on light intensity. Since the reactive side groups must be in close enough proximity for the interpolymer side chain reaction to occur, a threshold side chain density is required to form junction points. After extensive UV exposure, the elasticity of the network degraded, and the rate of degradation was faster for films polymerized at lower initial surface pressures.
- Published
- 2002
18. Point Mutagenesis and Cocrystallization of Wild-Type and Mutant Proteins: A Study of Solid-Phase Coexistence in Two-Dimensional Protein Arrays
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Sammy J. Farah, and Channing R. Robertson, Alice P. Gast, Szu-Wen Wang, and Wei-Hau Chang
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Streptavidin ,Mutant ,technology, industry, and agriculture ,Mutagenesis (molecular biology technique) ,Surfaces and Interfaces ,Condensed Matter Physics ,chemistry.chemical_compound ,Isoelectric point ,chemistry ,Biochemistry ,Phase (matter) ,Biotinylation ,Monolayer ,Electrochemistry ,Protein microarray ,Biophysics ,General Materials Science ,Spectroscopy - Abstract
We are studying the molecular organization of protein arrays using two-dimensional streptavidin crystals bound to biotinylated lipid monolayers at the air−water interface. We constructed a mutant form of the streptavidin protein that successfully alters the molecular organization of the streptavidin crystals. Cocrystallization of streptavidin carrying this single targeted point mutation with wild-type streptavidin yields two-dimensional crystals displaying a chiral morphology with molecular coexistence, indicating a solid-phase transition. The phase coexistence and resulting morphologies are reminiscent of two-dimensional crystal behavior of wild-type streptavidin near its isoelectric point, and this analogy is discussed. These results demonstrate the potential to manipulate protein array formation through point mutagenesis and cocrystallization.
- Published
- 2001
19. Role of N- and C-Terminal Amino Acids in Two-Dimensional Streptavidin Crystal Formation
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Channing R. Robertson, Szu Wen Wang, Sandy Koppenol, Patrick S. Stayton, Alice P. Gast, Viola Vogel, and Todd Edwards
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chemistry.chemical_classification ,In situ ,Streptavidin ,Phase transition ,Stereochemistry ,Mutant ,Surfaces and Interfaces ,Condensed Matter Physics ,Amino acid ,law.invention ,Crystal ,Crystallography ,chemistry.chemical_compound ,chemistry ,law ,Electrochemistry ,Recombinant DNA ,General Materials Science ,Crystallization ,Spectroscopy - Abstract
The carboxyl- and amino-terminal ends of streptavidin are near the site of protein−protein contacts in two-dimensional streptavidin crystals. The role of these C- and N-terminal residues in determining the pH-dependent phase behavior of crystallization has been investigated with site-directed truncation mutants. Commercial streptavidin (consisting primarily of amino acids 14−136) and two recombinant streptavidin forms, spanning residues 13−136 and 13−139, have been crystallized at pH 4−7. The commercial 14−136 protein crystallizes in three distinct lattice symmetries, P1, P2, and C222, respectively, depending on pH. The 13−136 mutant also crystallizes in three distinct lattices, but with a shifted pH profile that is attributed to the N-terminal residue. The presence of amino acids 137−139 inhibits the growth of crystals with P1 symmetry at low pH. In addition, we observe a solid−solid phase transition in situ from the P2 to the P1 crystal forms for the 13−136 recombinant protein at pH 5.2. We also demonst...
- Published
- 2000
20. Protease Activity on an Immobilized Substrate Modified by Polymers: Subtilisin BPN‘
- Author
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and Channing R. Robertson, Giuseppe Trigiante, Philip Frederick Brode, Alan R. Esker, Deborah S. Rauch, Alice P. Gast, Hyuk Yu, and Donn N. Rubingh
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Serine protease ,chemistry.chemical_classification ,Protease ,Ethylene oxide ,biology ,Chemistry ,medicine.medical_treatment ,Surfaces and Interfaces ,Polymer ,Condensed Matter Physics ,Hydrolysis ,chemistry.chemical_compound ,Adsorption ,Covalent bond ,Polymer chemistry ,Electrochemistry ,medicine ,biology.protein ,Copolymer ,Organic chemistry ,General Materials Science ,Spectroscopy - Abstract
We describe the adsorption and catalytic behavior of the serine protease subtilisin BPN‘ on controlled pore glass (CPG) beads with a short (aminopropyl) or a long (aminoalkyl CH2 > 12) chain covalent link separating the reporter peptide succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (sAAPFpNA) from the surface. The propyl-linked sAAPFpNA modified glass surface (aminopropyl CPG:sAAPFpNA) showed a 2-fold increase in protease adsorption over an aminopropyl−glass surface. In contrast, the sAAPFpNA surface with the long chain connector showed a 2-fold drop in adsorption relative to an aminoalkyl surface. BPN‘-catalyzed hydrolysis rates showed an inverse relationship to adsorption. Water-soluble polymers [poly(vinylpyrrolidone) (PVP), poly(ethylene oxide) (PEO), poly(4-vinylpyridine-N-oxide) (PVPO) and a copolymer of 1-vinyl-2-pyrrolidone and 1-vinylimidazole (PVPVI)] neutralize the 2-fold increase in BPN‘ adsorption and provide more than a 3-fold increase in the initial rate of hydrolysis for BP...
- Published
- 2000
21. Two-Dimensional Crystallization of Streptavidin Mutants
- Author
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Alice P. Gast, Channing R. Robertson, and Szu-Wen Wang
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Streptavidin ,chemistry.chemical_compound ,Crystallography ,Chemistry ,law ,Mutant ,Materials Chemistry ,Physical and Theoretical Chemistry ,Crystallization ,Protein crystallization ,Surfaces, Coatings and Films ,law.invention - Abstract
Many applications motivate investigations of the physical factors governing protein assembly in two dimensions, including two-dimensional (2D) protein crystallization for complex structural analyse...
- Published
- 1999
22. Pseudo First-Order Cleavage of an Immobilized Substrate by an Enzyme Undergoing Two-Dimensional Surface Diffusion
- Author
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Giuseppe Trigiante, Channing R. Robertson, and Alice P. Gast
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Surface diffusion ,Stereochemistry ,Chemistry ,Diffusion ,Substrate (chemistry) ,Photochemistry ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Biomaterials ,Chemical kinetics ,Colloid and Surface Chemistry ,Reaction rate constant ,Adsorption ,Extent of reaction ,Enzyme kinetics - Abstract
In this paper we study the reaction kinetics of an enzyme adsorbed on a peptide substrate surface. Although the adsorption is effectively irreversible, the enzyme is able to diffuse on the surface. Our reaction system consisted of the enzyme collagenase and the oligopeptide FALGPA, a substrate for the enzyme. A quartz surface was coated with covalently bound substrate molecules. The extent of reaction was monitored continuously in a flow cell via UV absorption. The data are compatible with a kinetic model based on a pseudo first-order diffusion/orientation rate-limiting step followed by a relatively fast chemical cleavage step. This model was validated by examining the pH dependence of the rate constant. Copyright 1999 Academic Press.
- Published
- 1999
23. An Interfacial Stress Rheometer To Study Rheological Transitions in Monolayers at the Air−Water Interface
- Author
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Curtis W. Frank, Gerald G. Fuller, Carlton F. Brooks, and Channing R. Robertson
- Subjects
Chemistry ,business.industry ,Rheometer ,Thermodynamics ,Surfaces and Interfaces ,Condensed Matter Physics ,Surface pressure ,Viscosity ,Optics ,Rheology ,Phase (matter) ,Dynamic modulus ,Monolayer ,Electrochemistry ,Newtonian fluid ,General Materials Science ,business ,Spectroscopy - Abstract
An interfacial stress rheometer has been constructed to study the rheology of Langmuir films subjected to time-dependent flows. A magnetized rod resides at the air−water interface and is set into oscillation by applying a sinusoidal magnetic field gradient. Analysis of the amplitude and phase of the resulting rod motion relative to the applied force allows the determination of the dynamic surface modulus, Gs*(ω), and measurement of the relative elastic and viscous contributions of the monolayer. Measurements at 22 °C were conducted on eicosanol (C20) and mixtures of a rigid-rod polymer, phthalocyaninatopolysiloxane (PcPS), dispersed in eicosanol. The surface pressure dependence of the rheology for eicosanol reveals the presence of a maximum in the loss modulus, Gs‘ ‘(ω), within the L2‘ phase at Π = 6 mN/m. In the LSI phase at pressures above 15 mN/m, the monolayer is Newtonian and has a surface viscosity of 0.03 mN·s/m. The mixtures of PcPS with eicosanol are known to have two-dimensional nematic behavior...
- Published
- 1999
24. Molecular Arrangement in Two-Dimensional Streptavidin Crystals
- Author
-
and Channing R. Robertson, Alice P. Gast, and Szu-Wen Wang
- Subjects
Streptavidin ,Morphology (linguistics) ,Chemistry ,Air water interface ,Surfaces and Interfaces ,Crystal structure ,Condensed Matter Physics ,law.invention ,Line defects ,chemistry.chemical_compound ,Crystallography ,Molecular level ,law ,Electrochemistry ,Molecule ,General Materials Science ,Crystallization ,Spectroscopy - Abstract
We studied the molecular arrangement of two-dimensional streptavidin crystals at the air−water interface over a range of pH values. We quantified the varying amounts of coexisting P1, P2, and C222 crystals in the different morphologies observed at pH 4.5−6.5. Chiral, needlelike crystals at pH 4.5 consist of P1 crystals with frequent line defects. Larger chiral domains near pH 5 are essentially all P1 coexisting with a small amount of P2, whereas at slightly higher pH values (near pH 5.5), H-shaped domains contain 4 times as much P1 coexisting with a P2/C222 mixture. Morphologies intermediate to these shapes exhibit intermediate compositions. Between pH ∼6−7, crystals all display a characteristic dendritic-X morphology, but arrangement at the molecular level is quite different compared with lower pH values. Crystals are mostly P2 in symmetry near pH 6, but at pH 7 and above, crystals have C222 symmetry. Coexistence of P2 and C222 crystals occurs at intermediate pH values. We determined the orientation and ...
- Published
- 1999
25. Orientation in a Fatty Acid Monolayer: Effect of Flow Type
- Author
-
T. Maruyama, Curtis W. Frank, Jörg Läuger, Gerald G. Fuller, and Channing R. Robertson
- Subjects
Brewster's angle ,Chemistry ,Mineralogy ,Surfaces and Interfaces ,Strain rate ,Plasticity ,Condensed Matter Physics ,Molecular physics ,Simple shear ,symbols.namesake ,Shear (geology) ,Monolayer ,Electrochemistry ,Fluid dynamics ,symbols ,General Materials Science ,Shear flow ,Spectroscopy - Abstract
The two-dimensional fluid dynamics of the different phases of a fatty acid monolayer (docosanoic acid) were examined. Using Brewster angle microscopy, we studied the polydomain structure of two liquid condensed phases (the L 2 and L' 2 phases) and the solid, S, phase in situ during the application of extensional flow and simple shear flow. We found that only the L2 phase deformed nearly reversibly with a liquid-like response. Nonsymmetric domain deformations were, however, found for that phase at the lowest rate of strain studied with a four-roll mill. At higher strain rates, plots of the evolution of strain against time collapsed onto a single curve so that the rate of strain was independent of roller speed. Furthermore, a critical strain y c exists above which the rate of strain shows a stepwise increase despite the constant velocity of the rollers. The two other phases, the L' 2 and S phases, experienced flow-induced reorientation of the lattice onto which the molecules are arranged. The reorientation process was accompanied by the appearance of shear bands in the monolayers at ±45° to the extension axis of both types of flow. The shear bands observed in the L' 2 phase were modeled as a plastic flow accompanying the molecular tilt reorientation developed within elastic regions of the monolayer. This model describes the time evolution of the bandwidth quite well and provides strong evidence of the existence of an additional phase within the conventional L' 2 phase region. In simple shear flow, the velocity profile for the L 2 phase across the gap of the shearing cell showed a nonlinear distribution of shear rates, which were highest at the center of the gap. Flow-induced breakup of domains was observed in the L' 2 phase subject to simple shear.
- Published
- 1998
26. Influence of pH on Two-Dimensional Streptavidin Crystals
- Author
-
Alice P. Gast, and Channing R. Robertson, and Michael T. Yatcilla
- Subjects
Streptavidin ,Intermolecular force ,Nucleation ,Crystal growth ,Surfaces and Interfaces ,Condensed Matter Physics ,law.invention ,Crystallography ,chemistry.chemical_compound ,chemistry ,law ,Biotinylation ,Electrochemistry ,General Materials Science ,Crystallization ,Protein crystallization ,Anisotropy ,Spectroscopy - Abstract
To obtain a general understanding of the effect of intermolecular interactions on the mechanisms of two-dimensional protein crystallization, we grow protein crystals and elicit a bulk molecular manipulation by changing system pH. Two-dimensional crystals of the bacterial protein streptavidin grown on a biotinylated lipid monolayer at an air−water interface, in the presence of the noncrystallizable impurity avidin, exhibit crystallographic and morphological changes as a function of subphase pH. Large two-dimensional crystalline arrays form within minutes across a pH range from 1.5 to 11. Crystals exhibit different pH-dependent structures, lattices with P1 symmetry for 1.5 < pH < 5, P1 and P2 lattices for 5 < pH < 6, and C222 lattices for 7 < pH < 11. P1 crystals nucleate rapidly and form thin needle-shaped crystals consistent with a strong growth anisotropy between the two crystallographic growth directions. C222 crystals grow more isotropically and exhibit H- and X-shapes. The nucleation rates and aspect ...
- Published
- 1998
27. Solid Phase Coexistence in Chiral Domains of Two-Dimensional Streptavidin Crystals
- Author
-
Claudia L. Poglitsch, Michael T. Yatcilla, Szu-Wen Wang, Alice P. Gast, and Channing R. Robertson
- Subjects
Streptavidin ,Crystal growth ,Surfaces and Interfaces ,Crystal structure ,Condensed Matter Physics ,Crystal ,chemistry.chemical_compound ,Crystallography ,chemistry ,Transmission electron microscopy ,Phase (matter) ,Electrochemistry ,Molecule ,General Materials Science ,Anisotropy ,Spectroscopy - Abstract
Growth of two-dimensional streptavidin crystals at the air−water interface has been used to study protein molecular interactions at various pH values. Between pH 5 and 6, several unique crystal domain shapes are visible with fluorescence microscopy. Characterization of a chiral domain with transmission electron microscopy reveals a composition of two coexisting crystal types. The bulk crystal, one previously seen at pH 4 (space group P1) (Hemming et al. J. Mol. Biol. 1995, 246, 308), has interspersed within it at approximately 5% aerial extent a new crystal type with unit cell parameters a = 116 A, b = 58 A, and γ = 107° (space group P2). This new form exists in the chiral domains as long narrow crystals and is characterized by relatively weak and anisotropic molecular interactions. The packing arrangement for the new P2 crystal at pH 5−6 exhibits characteristics of the pH 4 crystal and of the C222 crystal (Darst et al. Biophys. J. 1991, 59, 387) obtained at pH 7.
- Published
- 1997
28. In-situ studies of flow-induced phenomena in Langmuir monolayers
- Author
-
Gerhard Wegner, Takayuki Maruyama, Curtis W. Frank, Channing R. Robertson, A. Ferencz, Matthew C. Friedenberg, and Gerald G. Fuller
- Subjects
chemistry.chemical_classification ,Langmuir ,Chemistry ,business.industry ,Metals and Alloys ,Analytical chemistry ,Surfaces and Interfaces ,Polymer ,Stagnation point ,Linear dichroism ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Optics ,Monolayer ,Materials Chemistry ,Particle ,Thin film ,business ,Anisotropy - Abstract
Flow-induced orientation of the hairy-rod polymer poly(phthalocyaninato siloxane) (PcPS) dissolved at 5 mol.% in docosanoic acid and residing in a monolayer at the air-water interface is reported. The flow was induced through two routes. In one, a four-roll mill was immersed through the monolayer to produce a well-defined extensional flow. In the second, a Langmuir-Blodgett (LB) deposition process was carried out simultaneously onto three parallel, glass plates, thereby generating a stagnation point, with extensional flow between each pair of slides. Linear dichroism was used to obtain in situ the measurements of orientation in the Langmuir films themselves, and on the glass substrates. It was found that the flows generated by the LB process induced substantial orientation within the Langmuir film, and this anisotropy was also evident in the LB films that were deposited. The velocity fields were measured using a particle tracking technique and compared against the predictions of a model assuming that the glass substrates act as line sinks of material during the LB process. It was found that the meniscus region distorts this velocity profile and leads to decreased alignment of the polymer chains.
- Published
- 1996
29. In Situ Optical Studies of Flow-Induced Orientation in a Two-Dimensional Polymer Solution
- Author
-
Curtis W. Frank, Matthew C. Friedenberg, Channing R. Robertson, and Gerald G. Fuller
- Subjects
chemistry.chemical_classification ,Polymers and Plastics ,business.industry ,Arachidyl alcohol ,Organic Chemistry ,Relaxation (NMR) ,Two-dimensional polymer ,Polymer ,Inorganic Chemistry ,chemistry.chemical_compound ,Optics ,chemistry ,Flow (mathematics) ,Chemical physics ,Monolayer ,Materials Chemistry ,Perpendicular ,business ,Anisotropy - Abstract
The orientation dynamics of polymers in constrained geometries is considered through studies of monolayer films at the air−water interface. Here, in situ optical techniques are employed to probe flow orientation in monolayers of phthalocyaninatopolysiloxane dispersed in either docosanoic acid, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine, or arachidyl alcohol. Compression of the polymer monolayer creates alignment perpendicular to the compression direction. A well-defined extensional flow is imposed in the monolayer to study the dynamics of flow-induced anisotropy. The orientation process obeys a strain-scaling law, indicating the absence of relaxation on the time scale of the flow.
- Published
- 1996
30. Direct Visualization of Flow-Induced Anisotropy in a Fatty Acid Monolayer
- Author
-
Gerald G. Fuller, and Curtis W. Frank, Matthew C. Friedenberg, and Channing R. Robertson
- Subjects
Brewster's angle ,business.industry ,Chemistry ,Surfaces and Interfaces ,Condensed Matter Physics ,Surface pressure ,symbols.namesake ,Optics ,Flow (mathematics) ,Chemical physics ,Frequency domain ,Phase (matter) ,Microscopy ,Monolayer ,Electrochemistry ,symbols ,General Materials Science ,Elongation ,business ,Spectroscopy - Abstract
Brewster angle microscopy is used to directly visualize the influence of an applied extensional flow on the domain structure and molecular orientation of a docosanoic acid monolayer at the air−water interface. At a surface pressure of 12 mN/m and a subphase temperature of 15 °C (L2 phase), extensional flow causes domain elongation parallel to the extension axis. A frequency domain analysis of the Brewster angle images indicates that the domains undergo an affine deformation in response to flow. AT 20 mN/m (L2‘ phase), the flow modifies not only the domain structure of the monolayer but also the azimuthal orientation of the fatty acid molecules. This flow-alignment process is strain-rate dependent. Thus, flow can couple to the monolayer order over a variety of length scales.
- Published
- 1996
31. Deformation and Relaxation Processes of Mono- and Bilayer Domains of Liquid Crystalline Langmuir Films on Water
- Author
-
Gerald G. Fuller, Jörg Läuger, Channing R. Robertson, and Curtis W. Frank
- Subjects
Brewster's angle ,business.industry ,Chemistry ,Bilayer ,Surfaces and Interfaces ,Deformation (meteorology) ,Condensed Matter Physics ,Breakup ,Thermotropic crystal ,symbols.namesake ,Optics ,Chemical physics ,Liquid crystal ,Monolayer ,Electrochemistry ,symbols ,Relaxation (physics) ,General Materials Science ,business ,Spectroscopy - Abstract
Flow deformation and relaxation of mono- and bilayer domains of a thermotropic liquid crystal at the air−water interface have been studied by means of simultaneous application of extensional flow fields and Brewster angle microscopy measurements. In an extentional flow, mono- and bilayer domains are found to be distorted into long, stringlike domains that are stable. This is due to the absence of Rayleigh instabilities in the two-dimensional case, which otherwise lead to the occurrence of a break-up mechanism in three dimensions. However, small fluctuations in the domain thickness lead to defects that can cause breakup of the string domains followed by a retraction of the two domain ends due to line tension effects. In relaxation experiments covering both the high deformation bola-shaped regime and the small-deformation regime of slightly deformed domains, the line tensions of mono- and bilayer domains were determined. Both deformation regimes yielded the same values for the line tension of the mono- and ...
- Published
- 1996
32. Enzymes on Immobilized Substrate Surfaces: Reaction
- Author
-
Pamela B. Gaspers, Channing R. Robertson, and Alice P. Gast
- Subjects
Surface diffusion ,Stereochemistry ,Chemistry ,Diffusion ,Kinetics ,Substrate (chemistry) ,Concentration effect ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Biomaterials ,Reaction rate ,Colloid and Surface Chemistry ,Chemical engineering ,Microbial collagenase ,Enzyme kinetics - Abstract
The goal of this paper is to measure the overall reaction rate of irreversibly adsorbed but mobile enzymes on substrate-coated surfaces and to model the interplay between reaction and diffusion in this system. Our model system uses collagenase, an enzyme that degrades native collagen, and synthetic surfaces covered with a peptide that is a substrate for collagenase. We employ a multisurface flow cell device to measure the change of ultraviolet absorbance as collagenase hydrolyzes the surface-bound peptide substrate. The overall reaction rate is enhanced as the surface enzyme concentration is increased. By changing the buffer pH we discern the influence of the intrinsic kinetic rate on the overall reaction rate. Employing surfaces containing fewer bound substrates, we probe the effect of the distance between substrates on the overall reaction rate to show that this process is governed by both reaction and diffusion. Using a modified Smoluchowski theory, we model this interplay to determine the intrinsic reaction rate of the surface process and compare this value to those found for collagenase reaction in solution.
- Published
- 1995
33. Enzymes on Immobilized Substrate Surfaces: Diffusion
- Author
-
Alice P. Gast, Channing R. Robertson, and Pamela B. Gaspers
- Subjects
chemistry.chemical_classification ,Chromatography ,Immobilized enzyme ,Diffusion ,Substrate (chemistry) ,Concentration effect ,Surfaces and Interfaces ,Condensed Matter Physics ,Adsorption ,Enzyme ,chemistry ,Chemical engineering ,Electrochemistry ,General Materials Science ,Spectroscopy - Published
- 1994
34. Formation of Bilayer Disks and Two-Dimensional Foams on a Collapsing/Expanding Liquid-Crystal Monolayer
- Author
-
Matthew C. Friedenberg, Curtis W. Frank, Gerald G. Fuller, and Channing R. Robertson
- Subjects
Brewster's angle ,Nitrile ,Chemistry ,Air water interface ,business.industry ,Bilayer ,Collapse (topology) ,Surfaces and Interfaces ,Condensed Matter Physics ,symbols.namesake ,chemistry.chemical_compound ,Optics ,Liquid crystal ,Chemical physics ,Monolayer ,Microscopy ,Electrochemistry ,symbols ,General Materials Science ,business ,Spectroscopy - Published
- 1994
35. Molecular analysis of two-dimensional protein crystallization
- Author
-
Channing R. Robertson, Seth A. Darst, Roger D. Kornberg, Andrew C. Ku, and Alice P. Gast
- Subjects
Streptavidin ,Morphology (linguistics) ,Chemistry ,Intermolecular force ,General Engineering ,law.invention ,Molecular analysis ,chemistry.chemical_compound ,Crystallography ,law ,Microscopy ,Molecule ,Physical and Theoretical Chemistry ,Electron microscope ,Protein crystallization - Abstract
Two-dimensional crystals of streptavidin formed on lipid layers at the air-water interface were cross-linked to preserve their structure during transfer to a solid support. The morphology of the crystals viewed at the air-water interface by light microscopy could then be related to the molecular structure revealed in the electron microscope. The preferred growth direction of the crystals proved to be along one of two intersecting rows of intermolecular contacts. A secondary direction arises during a transition to dendritic growth
- Published
- 1993
36. Interfacial biocatalysis on charged and immobilized substrates: the roles of enzyme and substrate surface charge
- Author
-
Curtis W. Frank, Luis G. Cascao-Pereira, Bob E. Feller, Channing R. Robertson, and James T. Kellis
- Subjects
Models, Molecular ,Protein Conformation ,Surface Properties ,Diffusion ,Static Electricity ,Ionic bonding ,Reaction rate ,Adsorption ,Static electricity ,Electrochemistry ,Organic chemistry ,Animals ,Humans ,General Materials Science ,Surface charge ,Spectroscopy ,Cellulomonas ,Chemistry ,Osmolar Concentration ,Serum Albumin, Bovine ,Surfaces and Interfaces ,Hydrogen-Ion Concentration ,Condensed Matter Physics ,Electrostatics ,Enzymes, Immobilized ,Kinetics ,Chemical engineering ,Ionic strength ,Biocatalysis ,Cattle ,Serine Proteases - Abstract
An enzyme charge ladder was used to examine the role of electrostatic interactions involved in biocatalysis at the solid-liquid interface. The reactive substrate consisted of an immobilized bovine serum albumin (BSA) multilayer prepared using a layer-by-layer technique. The zeta potential of the BSA substrate and each enzyme variant was measured to determine the absolute charge in solution. Enzyme adsorption and the rate of substrate surface hydrolysis were monitored for the enzyme charge ladder series to provide information regarding the strength of the enzyme-substrate interaction and the rate of interfacial biocatalysis. First, each variant of the charge ladder was examined at pH 8 for various solution ionic strengths. We found that for positively charged variants the adsorption increased with the magnitude of the charge until the surface became saturated. For higher ionic strength solutions, a greater positive enzyme charge was required to induce adsorption. Interestingly, the maximum catalytic rate was not achieved at enzyme saturation but at an invariable intermediate level of adsorption for each ionic strength value. Furthermore, the maximum achievable reaction rate for the charge ladder was larger for higher ionic strength values. We propose that diffusion plays an important role in interfacial biocatalysis, and for strong enzyme-substrate interaction, the rate of diffusion is reduced, leading to a decrease in the overall reaction rate. We investigated the effect of substrate charge by varying the solution pH from 6.1 to 8.7 and by examining multiple ionic strength values for each pH. The same intermediate level of adsorption was found to maximize the overall reaction rate. However, the ionic strength response of the maximum achievable rate was clearly dependent on the pH of the experiment. We propose that this observation is not a direct effect of pH but is caused by the change in substrate surface charge induced by changing the pH. To prove this hypothesis, BSA substrates were chemically modified to reduce the magnitude of the negative charge at pH 8. Chemical modification was accomplished by the amidation of aspartic and glutamic acids to asparagine and glutamine. The ionic strength response of the chemically modified substrate was considerably different than that for the native BSA substrate at an identical pH, consistent with the trend based on substrate surface charge. Consequently, for substrates with a low net surface charge, the maximum achievable catalytic rate of the charge ladder was relatively independent of the solution ionic strength over the range examined; however, at high net substrate surface charge, the maximum rate showed a considerable ionic strength dependence.
- Published
- 2010
37. In their own words: an epoch of deceit and deception
- Author
-
Richard D. Hurt and Channing R. Robertson
- Subjects
Nicotine ,Tobacco harm reduction ,Cigarette smoking ,Addiction ,media_common.quotation_subject ,medicine ,EPOCH (chemotherapy) ,Deception ,Psychology ,Tobacco industry ,Social psychology ,medicine.drug ,media_common - Published
- 2010
38. Dendritic growth of two-dimensional protein crystals
- Author
-
Seth A. Darst, Roger D. Kornberg, Andrew C. Ku, Channing R. Robertson, and Alice P. Gast
- Subjects
Crystallography ,Chemistry ,Monolayer ,Electrochemistry ,Fluorescence microscope ,General Materials Science ,Crystal growth ,Surfaces and Interfaces ,Condensed Matter Physics ,Protein crystallization ,Crystal morphology ,Spectroscopy - Published
- 1992
39. Use of glucose starvation to limit growth and induce protein production inEscherichia coli
- Author
-
Serena Schippa, Joseph R. Tunner, Channing R. Robertson, and Abdul Matin
- Subjects
Cell growth ,Operon ,Metabolite ,lac operon ,Bioengineering ,Chemostat ,Biology ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,law.invention ,chemistry.chemical_compound ,chemistry ,Biochemistry ,law ,Recombinant DNA ,medicine ,Energy source ,Escherichia coli ,Biotechnology - Abstract
The use of glucose starvation to uncouple the production of recombinant beta-galactosidase from cell growth in Escherichia coli was investigated. A lacZ operon fusion to the carbon starvation-inducible cst-1 locus was used to control beta-galactosidase synthesis. beta-Galactosidase induction was observed only under aerobic starvation conditions, and its expression continued for 6 h following the onset of glucose starvation. The cessation of beta-galactosidase expression closely correlated with the exhaustion of acetate, an overflow metabolite of glucose, from the culture medium. Our results suggest the primary role of acetate in cst-1-controlled protein expression is that of an energy source. Using this information, we metered acetate to a glucose-starved culture and produced a metabolically sluggish state, where growth was limited to a low linear rate and production of recombinant beta-galactosidase occurred continuously throughout the experiment. The cst-1 controlled beta-galactosidase synthesis was also induced at low dilution rates in a glucose-limited chemostat, suggesting possible applications to high-density cell systems such as glucose-limited recycle reactors. This work demonstrates that by using an appropriate promoter system and nutrient limitation, growth can be restrained while recombinant protein production is induced and maintained.
- Published
- 1992
40. Open doorway to truth: legacy of the Minnesota tobacco trial
- Author
-
Monique E Muggli, Channing R. Robertson, Nikki J. Lockhart, Richard D. Hurt, and Jon O. Ebbert
- Subjects
medicine.medical_specialty ,Minnesota ,Information Storage and Retrieval ,Tobacco Industry ,Disclosure ,Documentation ,Review ,Public administration ,World Health Organization ,Tobacco industry ,Medicine ,Humans ,Complicity ,Social Responsibility ,business.industry ,Public health ,Liability ,Tobacco control ,Smoking ,Liability, Legal ,General Medicine ,business ,Settlement (litigation) ,Social responsibility - Abstract
More than a decade has passed since the conclusion of the Minnesota tobacco trial and the signing of the Master Settlement Agreement (MSA) by 46 US State Attorneys General and the US tobacco industry. The Minnesota settlement exposed the tobacco industry's long history of deceptive marketing, advertising, and research and ultimately forced the industry to change its business practices. The provisions for public document disclosure that were included in the Minnesota settlement and the MSA have resulted in the release of approximately 70 million pages of documents and nearly 20,000 other media materials. No comparable dynamic, voluminous, and contemporaneous document archive exists. Only a few single events in the history of public health have had as dramatic an effect on tobacco control as the public release of the tobacco industry's previously secret internal documents. This review highlights the genesis of the release of these documents, the history of the document depositories created by the Minnesota settlement, the scientific and policy output based on the documents, and the use of the documents in furthering global public health strategies.
- Published
- 2009
41. Manipulation of hydrophobic interactions in protein adsorption
- Author
-
Robert D. Tilton, Alice P. Gast, and Channing R. Robertson
- Subjects
Hydrophobic effect ,chemistry.chemical_classification ,Adsorption ,Enzyme ,chemistry ,Electrochemistry ,Organic chemistry ,General Materials Science ,Surfaces and Interfaces ,Condensed Matter Physics ,Spectroscopy ,Protein adsorption - Published
- 1991
42. Characterization of immobilized cell growth rates using autoradiography
- Author
-
Channing R. Robertson, Philip S. Stewart, and Steven F. Karel
- Subjects
Chromatography ,Pulse labelling ,Cell growth ,chemistry.chemical_element ,Bioengineering ,Microporous material ,Biology ,Applied Microbiology and Biotechnology ,Membrane ,chemistry ,Biochemistry ,Emulsion ,Growth rate ,Energy source ,Carbon ,Biotechnology - Abstract
The growth of immobilized Escherichia coli was analyzed by pulse-chase radioisotope labeling of the cell mass with (35)SO(4) (2-) and subsequent liquid emulsion autoradiography of thin cross sections of the cell aggregate. Bacteria were retained in a planar aggregate on a microporous membrane and grown anaerobically on a phosphate-buffered medium with glucose as the sole carbon and energy source. A mathematical model of immobilized cell growth and convection was used to predict the distribution of label in the cell mass and permit information about both the magnitude and variation in the intrinsic growth rate to be extracted. Growing zone dimensions ranging from 4 to 48 mum and growth rates from 0.28 to 0.5 h(-1) were found. Data collected at low glucose concentrations were consistent with a zero-order description of intrinsic growth kinetics. At high glucose concentrations, conditions under which the system was subject to significant pH inhibition, the data were best described by the prediction of a first-order kinetic model. When coupled with a suitable analytical framework, the combination of radioisotope labeling and autoradiography provides a general method for characterizing immobilized cell growth rates.
- Published
- 1991
43. Hydraulic Permeability of Immobilized Bacterial Cell Aggregates
- Author
-
Channing R. Robertson and Jeffrey D. Fowler
- Subjects
Convection ,Materials science ,Ecology ,Membrane reactor ,Physiology and Biotechnology ,Applied Microbiology and Biotechnology ,law.invention ,Volumetric flow rate ,Permeability (earth sciences) ,Membrane ,Hydraulic conductivity ,law ,Volume fraction ,Biophysics ,Electron microscope ,Food Science ,Biotechnology - Abstract
A dense aggregate of cells was retained in a reactor by a supported porous membrane. A continuous flow of nutrient medium was maintained through the cell aggregate and membrane. The hydraulic resistance of the cell aggregate was monitored throughout experiments with either growing or chemically cross-linked cells, under conditions of varying flow rates. Digital image analysis was used to characterize the sizes, separations, and orientations of several thousand individual cells in electron micrographs of chemically cross-linked cell aggregates. Two nonlinear phenomena were observed. First, the hydraulic resistance varied in direct relation to and reversibly with flow rate. Second, in constant flow-rate experiments the hydraulic resistance increased with time at a faster rate than could be attributed to cell growth. Both of these phenomena were dependent upon and could be explained by the ability of cells to move with respect to one another, under the influences of Brownian motion and of convection. Such relative motion could allow changes in net alignment of cells in the direction of flow and in the volume fraction of cells in the aggregate. This explanation is consistent with image analysis data. The observed sensitivity of hydraulic resistance to flow rate was inconsistent with a model that assumed elastic deformation of individual cells, and no evidence of cell deformation was found in electron micrographs.
- Published
- 1991
44. Metabolic behavior of immobilized aggregates of Escherichia coli under conditions of varying mechanical stress
- Author
-
Channing R. Robertson and J. D. Fowler
- Subjects
Yield (engineering) ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Exponential growth ,Escherichia coli ,medicine ,Bioreactor ,Anaerobiosis ,Ecology ,biology ,Catabolism ,Metabolism ,Water-Electrolyte Balance ,biology.organism_classification ,Glucose ,Biochemistry ,Fermentation ,Biophysics ,Stress, Mechanical ,Acids ,Bacteria ,Research Article ,Food Science ,Biotechnology - Abstract
Experiments were conducted on immobilized aggregates of Escherichia coli cells. Mechanical stress was applied by forcing a convective stream of nutrient medium through the aggregate. It was shown to be possible to maintain uniform exponential growth with this convective supply of nutrients. Analysis of effluent from the system allowed investigation of metabolic responses unambiguously attributable to mechanical stress. A reversible increase in catabolic activity was observed after an increase in mechanical stress. Changes in the level of catabolism were accompanied by an alteration in the total acid yield on glucose and in the spectrum of organic acids produced during glucose fermentation. The behavior observed here was likely due to an osmoregulatory response induced by the mechanically stressed bacteria to counteract changes in shape.
- Published
- 1991
45. Waking a sleeping giant: the tobacco industry's response to the polonium-210 issue
- Author
-
Monique E Muggli, Channing R. Robertson, Richard D. Hurt, and Jon O. Ebbert
- Subjects
Polonium ,medicine.medical_specialty ,chemistry.chemical_element ,Tobacco Industry ,Tobacco industry ,Tobacco smoke ,Public Relations ,Dissolvable tobacco ,Tobacco in Alabama ,Environmental health ,Research Support as Topic ,Tobacco ,Medicine ,Humans ,education ,Tobacco harm reduction ,Publishing ,education.field_of_study ,Inhalation Exposure ,business.industry ,Information Dissemination ,Public health ,Framing Health Matters ,Smoking ,Public Health, Environmental and Occupational Health ,Organizational Policy ,United States ,chemistry ,Air Pollutants, Radioactive ,Consumer Product Safety ,Warning label ,Tobacco Smoke Pollution ,business - Abstract
The major tobacco manufacturers discovered that polonium was part of tobacco and tobacco smoke more than 40 years ago and attempted, but failed, to remove this radioactive substance from their products. Internal tobacco industry documents reveal that the companies suppressed publication of their own internal research to avoid heightening the public’s awareness of radioactivity in cigarettes. Tobacco companies continue to minimize their knowledge about polonium-210 in cigarettes in smoking and health litigation. Cigarette packs should carry a radiation-exposure warning label.
- Published
- 2008
46. Diffusional limitations of immobilizedEscherichia coli in hollow-fiber reactors: Influence on31P NMR spectroscopy
- Author
-
Steven F. Karel, Channing R. Robertson, and Catherine A. Briasco
- Subjects
chemistry.chemical_classification ,Sugar phosphates ,Chemistry ,Intracellular pH ,Diffusion ,Analytical chemistry ,Bioengineering ,Nuclear magnetic resonance spectroscopy ,equipment and supplies ,Applied Microbiology and Biotechnology ,Reaction rate ,NMR spectra database ,Transmission electron microscopy ,Mass transfer ,Biotechnology - Abstract
Escherichia coli cells were immobilized and grown in hollow-fiber reactors allowing simultaneous NMR spectroscopy and perfusion with nutrient medium. The extent to which the cells were starved due to inadequate mass transfer was predicted using a mathematical model of reaction and diffusion. Reactors were experimentally characterized using (35)S autoradiography to visualize spatial variations in protein synthesis rates and transmission electron microscopy to indicate spatial variations in cell morphology. Mass transfer limitations in reactors operated at 37 degrees C were shown to be severe, with regions of starved cells occupying up to 80% of the cell-containing region. Phosphorus-31 nuclear magnetic resonance (NMR) spectra of the immobilized, perfused cells revealed abnormally low volume-averaged concentrations of sugar phosphates, NTP, and ratios of NTP/NDP in these reactors. Intracellular pH was also depressed in the cells. In order to overcome mass transfer limitations in the cell layer, the reactor growth temperature was decreased. Sulfur-35 autoradiographs of a reactor operated at 16 degrees C did not indicate the presence of starved cells. The NMR spectra obtained from this reactor showed near-normal intracellular pH, metabolite concentrations, and NTP/NDP ratios. The presence of significant mass transfer limitations in a perfused cell sample during NMR spectroscopy is generally undesirable since the resulting spectra can be ambiguous and difficult to interpret. The strategy adopted in this work, namely estimation of the relative rates of reaction and diffusion in the cell mass and appropriate changes in reactor design and operating parameters, should prove generally applicable for the design of perfused cell samples for NMR spectroscopic experiments.
- Published
- 1990
47. Surface diffusion of interacting proteins. Effect of concentration on the lateral mobility of adsorbed bovine serum albumin
- Author
-
Alice P. Gast, Robert D. Tilton, and Channing R. Robertson
- Subjects
Self-diffusion ,Surface Properties ,Diffusion ,Serum albumin ,Analytical chemistry ,Biophysics ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Biophysical Phenomena ,Adsorption ,Methylmethacrylates ,Bovine serum albumin ,Surface diffusion ,Aqueous solution ,biology ,Chemistry ,Serum Albumin, Bovine ,021001 nanoscience & nanotechnology ,Photobleaching ,0104 chemical sciences ,Models, Chemical ,biology.protein ,0210 nano-technology ,Research Article - Abstract
Surface diffusion of bovine serum albumin absorbed from aqueous solution to poly(methylmethacrylate) surfaces is significantly hindered by protein-protein lateral interactions. The long-time self diffusion coefficient measured by fluorescence recovery after pattern photobleaching decreases by approximately one order of magnitude as the surface area fraction occupied by protein increases from 0.10 to 0.69. Qualitative features of the surface concentration dependence of the self diffusion coefficient can be described by several recent models for lateral diffusion of interacting species. The mobile fraction is independent of the surface concentration, and both the self diffusion coefficient and the mobile fraction are constant between 15 min and 7 h of adsorption.
- Published
- 1990
- Full Text
- View/download PDF
48. Renal medullary microcirculation
- Author
-
Thomas L. Pallone, Channing R. Robertson, and Rex L. Jamison
- Subjects
Kidney Medulla ,Kidney ,Measurement method ,Medullary cavity ,Physiology ,business.industry ,Microcirculation ,Urinary system ,Hemodynamics ,General Medicine ,Anatomy ,medicine.anatomical_structure ,Physiology (medical) ,medicine ,Renal medulla ,Animals ,Humans ,business ,Molecular Biology - Abstract
Article de synthese sur la microcirculation dans la partie medullaire du rein chez les mammiferes. Anatomie; modeles de microcirculation; hematocrite; techniques, mesures et regulation du debit sanguin medullaire
- Published
- 1990
49. Lateral diffusion of bovine serum albumin adsorbed at the solid-liquid interface
- Author
-
Robert D. Tilton, Channing R. Robertson, and Alice P. Gast
- Subjects
Surface diffusion ,education.field_of_study ,Total internal reflection ,Total internal reflection fluorescence microscope ,Eosin ,biology ,Chemistry ,Population ,Fluorescence spectrometry ,Analytical chemistry ,Photobleaching ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Biomaterials ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,biology.protein ,Bovine serum albumin ,education - Abstract
The lateral mobility of eosin isothiocyanate-labeled bovine serum albumin irreversibly adsorbed to poly(methylmethacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS) surfaces from aqueous solution was measured by a combination of total internal reflection fluorescence and fluorescence recovery after pattern photobleaching techniques. Lateral mobility over distances of several micrometers was probed by photobleaching and monitoring fluorescence with the fringe pattern formed by two intersecting coherent laser beams in total internal reflection. The period of the fringes determined the characteristic length for transport on the surface and was varied by changing the angle of intersection. The dependence of the fluorescence recovery time on the period of the fringes indicates that lateral migration of the adsorbed protein on PMMA is by surface diffusion with D = (1.2 ± 0.3) × 10−9cm2/s, while the extent of fluorescence recovery reveals the coexistence of a mobile population and a population that is apparently immobile over 20 min. For comparison, D = (2.6 ± 0.1) × 10−9cm2/s on PDMS.
- Published
- 1990
50. Nutrient Transport and Cellular Morphology in Immobilized Cell Aggregates
- Author
-
J. D. Fowler and Channing R. Robertson
- Subjects
Bacteriological Techniques ,Magnetic Resonance Spectroscopy ,Chemistry ,General Neuroscience ,Diffusion ,Cell ,Nuclear magnetic resonance spectroscopy ,Models, Theoretical ,medicine.disease_cause ,Bacterial Adhesion ,General Biochemistry, Genetics and Molecular Biology ,Microscopy, Electron ,medicine.anatomical_structure ,Nutrient ,History and Philosophy of Science ,Biochemistry ,Escherichia coli ,medicine ,Autoradiography ,Cellular Morphology - Published
- 1990
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