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Inhaltsverzeichnis

Frontmatter

Analysis of the Adsorption Kinetics

1. Protein Adsorption Kinetics: Influence of Substrate Electric Potential

Abstract
Substrate electric potential plays an important role in determining the adsorptive behavior of proteins and other macromolecules. In this chapter we describe the measurement of protein adsorption kinetics in the presence of an applied potential using optical waveguide lightmode spectroscopy. We analyze the resulting kinetics in terms of transport- and surface-limited models and show that while substrate potential is an important influencing factor, transport limited by convective diffusion and adsorption in seeming violation of electrostatic principles are prevalent in simple protein systems.
Paul R. Van Tassel

2. From Kinetics to Structure: High Resolution Molecular Microscopy

Abstract
Existing methods of protein structural determination either present a static picture under unnatural conditions or impose a considerable perturbation on the molecule during measurement. This chapter introduces high resolution optical waveguide lightmode spectroscopy (OWLS) as an alternative non-imaging technique of great power and versatility enabling the structure and dynamics of proteins at interfaces to be characterized with unprecedented richness and precision.
Jeremy J. Ramsden

3. Initial Adsorption Kinetics in a Rectangular Thin Channel, and Coverage-Dependent Structural Transition Observed by Streaming Potential

Abstract
One fundamental parameter contributing to the analysis of protein-solid surface interactions is the adsorption kinetic constant k a. For a rectangular channel we give a global representation of the initial experimental adsorption constant k at distance x from the entrance channel, as a function of wall shear rate γ, through the variable 1.86 k(x/γ)1/3. It is possible to visualize on a single graph the adsorption kinetic constant, the diffusion coefficient, depletion magnitude at the interface, and its thickness relative to that of the transport-limited Lévêque limit. With radiolabeled molecules (with a γ emitter like 125I) and well-defined geometries, calibration does not require a known solute diffusion coefficient and is obtained fromthe abrupt variation of radioactivity at the buffer-protein solution change and vice-versa. Experimental data obtained for the system ±-chymotrypsin/mica show that when surface coverage reaches some critical level, the streaming potential becomes almost independent of further interfacial concentration increase; this suggests an interfacial structural transition induced by interactions between adsorbed molecules. Several models based on protein-protein dipolar interactions are proposed.
Philippe Déjardin, Elena N. Vasina

Analysis of the Structure at the Interface

4. Dual Polarisation Interferometry: An Optical Technique to Measure the Orientation and Structure of Proteins at the Solid-Liquid Interface in Real Time

Abstract
The study of the orientation, structure, and function of proteins at interfaces in real time is demanding and complex in nature. For the last 40 years the primary means for the study of protein structure has been x-ray crystallography. This is, of course, a solid-state technique and not real time in nature. Over the last 20 years several techniques to address, at least in part, this important area have been adopted. These range from biosensors, which measure the rates of change of processes at interfaces, to analytical techniques such as neutron reflection, which are capable of providing detailed information on (protein) layer structures at interfaces. In this article a new analytical technique is described, dual polarisation interferometry (DPI), which combines the analytical nature of neutron reflection techniques with the real-time, bench-top accessibility associated with biosensors. Several examples of the measurement of protein orientation, structure and function are presented to demonstrate the value of the technique. A description of the fundamental physical principles of the measurement and verification of the technique are also provided.
Neville Freeman

5. Total Internal Reflection Ellipsometry: Monitoring of Proteins on Thin Metal Films

Abstract
A measurement technique based on ellipsometry performed under conditions of total internal reflection is presented here. This technique is called total internal reflection ellipsometry (TIRE). When extended with the surface plasmon resonance effect, TIRE becomes a powerful tool for monitoring protein adsorption on thin metal films. A brief description of TIRE is presented here together with some examples of measurement system setups. Two examples of applications are included, followed by a short presentation of possible future applications of TIRE.
Michal Poksinski, Hans Arwin

6. Conformations of Proteins Adsorbed at Liquid-Solid Interfaces

Abstract
Nonspecific adsorption of globular proteins induces conformational changes that depend upon both on the nature of the sorbent phase and on the structural stability of the protein. Because proteins can adopt various flexible three-dimensional structures under external perturbation, the surface contact with a sorbent phase can stabilise or not different conformers, which will influence the adsoprtion and desorption kinetics. Biophysical techniques such as Fourier transform infrared (FTIR) and circular dichroism spectroscopies enable the determination of the extent in secondary structures of the adsorbed protein at the aqueous-solid interface. Based on practical viewpoints of protein adsorption, we review the findings obtained in a wide range of sorbent phases such as mineral particles, colloidal systems, planar surfaces chemically modified by polymers, or self-assembled monolayers. We lay emphasis on the importance of obtaining insights into both structure and solvation during adsorption by the combinedNH/NDisotope exchange and attenuated total reflectance (ATR)-FTIR techniques. We present illustrative cases of adsorption of proteins of low and high structural stabilities, bovine serum albumin and lysozyme, respectively, on either hydrophobic or hydrophilic supports.
Sylvie Noinville, Madeleine Revault

7. Evaluation of Proteins on Bio-Devices

Abstract
The evaluation of proteins on bio-devices is one of the most important issues in biotechnology fields. In particular, evaluation of the changes in orientation and partial changes in structure of immobilized proteins are required for the development of sophisticated bio-devices. There are various methods being developed for protein evaluation. In this chapter, time-of-flight secondary ion mass spectrometry (TOF-SIMS), one of the most sensitive surface analysis methods, is described in terms of protein measurement. TOF-SIMS is useful for the evaluation of bio-device surfaces because it provides a submicron-scale mapping, orientation, and conformation of immobilized proteins on devices. In addition, TOF-SIMS requires no pretreatment of samples, such as labeling with a fluorescent probe or coating with metallic thin films, to prepare the samples. In the near future, conformation change of proteins after reactions or environmental change, such as changes in pH and orientation of binding sites of immobilized protein, will be measured with TOF-SIMS based on the analysis of partial chemical structures.
Satoka Aoyagi, Masahiro Kudo

Some Applications

8. Fibronectin at Polymer Surfaces with Graduated Characteristics

Abstract
Globular proteins adsorbed onto artificialmaterials often exhibit different functional characteristics due to an altered availability formolecular interactions. This effect is caused by the patterns of substrate-protein interactions and is attributable to conformational changes as well as to the orientation and/or the anchorage of the surface-confined proteins. To highlight this interrelation we report a detailed experimental investigation of the adsorption and displacement of fibronectin, a key protein of the extracellular matrix that enables cell adhesion, at a set of polymer thin films with various hydrophilicity, charge density and swelling characteristics. The patterns of protein displacement were analysed quantitatively for several substrates and adsorbed protein amounts, referring to a model recently suggested by Huetz et al. (Langmuir 11:3145–3152, 1995). The patterns of protein displacement were related to the substrate characteristics and the conditions applied during the formation of the protein layer (i. e. the solution concentration). These findings were compared with the reorganisation of adsorbed fibronectin on the compared polymer substrates by endothelial cells in culture. The results demonstrate that a certain binding strength of fibronectin is required to support the cell-driven formation of fibronectin fibrils, which, in turn, is an important prerequisite for the differentiation of the cells.
Tilo Pompe, Lars Renner, Carsten Werner

9. Development of Chemical Microreactors by Enzyme Immobilization onto Textiles

Abstract
Advanced biotechnological techniques are now being used in the chemical engineering of membrane processes, notably enzyme immobilization procedures, biosensors, and, more recently, proteomics. The knowledge and increasingly fine control of the production and reactivity of enzymes also profits research whose aim is to use on a large scale the catalytic material properties of biocatalysts (enzymes). These materials with reactive properties could be introduced into membrane technology, opening as a new field the treatment of liquid media according to the concept of membrane bioreactors, where the membrane itself acts as the chemical reactor. As will be discussed herein, the ionic-exchanging textiles will be involved in this futurology.
Christophe Innocent, Patrick Seta

10. Approaches to Protein Resistance on the Polyacrylonitrile-based Membrane Surface: an Overview

Abstract
Protein adsorption and/or deposition at the surface of polymeric membranes play important roles in membrane separation processes. Although polyacrylonitrile-based membranes have been used successfully in many fields, surface modifications to improve the protein resistance and hemocompatibility have received considerable interest. Various methods such as copolymerization, grafting, physical adsorption, biomacromolecule immobilization, and biomimetic modification, have been explored to build a friendly microenvironment for proteins, especially enzymes, at the membrane surface. Herein, the behaviors of proteins at the surfaces of polyacrylonitrile-based membranes are reviewed.
Ling-Shu Wan, Zhi-Kang Xu, Xiao-Jun Huang

11. Modulation of the Adsorption and Activity of Protein/Enzyme on the Polypropylene Microporous Membrane Surface by Surface Modification

Abstract
As an excellent membrane material, polypropylene microporous membrane (PPMM), has received much consideration in recent years. PPMM has also been used widely in many fields such as water treatment, enzyme immobilization, and blood oxygenation. However, poor surface properties cumber the further applications of this material. Many efforts have been made to solve this problem, and surface modification seems to be the most efficient way. In this article we introduce some work in which surface modification was conducted to reduce nonspecific protein adsorption and to modulate the activity of immobilized enzymes on the PPMM surface. We focus on the studies of our group, although other researches are also discussed.
Qian Yang, Zhi-Kang Xu, Zheng-Wei Dai

12. Nonbiofouling Surfaces Generated from Phosphorylcholine-Bearing Polymers

Abstract
The preparation and characterization of nonbiofouling surfaces generated from phosphorylcholine (PC)-bearing polymers are described. It is proposed that PC groups are an optimum surface with which to create biointerfaces because the surfaces have similarities with biomembranes. Nonspecific protein adsorption is generally the first process to occur when polymers come into contact with the vital environment, and this induces unfavorable bioreactions. Due to the properties of PC surfaces, such as high hydrophilicity, water structure, and zero )-potential, protein adsorption is effectively reduced on the PC-bearing surface. This surface property may be important for biomedical applications. The well-defined design of PC surfaces is also introduced in this chapter. These surfaces may be micro-or nanofabricated for, for example, medical devices and sensors. Control of cell-material interactions is effective on PC-bearing surfaces due to the negligible nonspecific interactions.
Yasuhiko Iwasaki, Nobuo Nakabayashi, Kazuhiko Ishihara

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