Article

In-depth characterization and computational 3D reconstruction of flagellar filament protein layer structure based on in situ spectroscopic ellipsometry measurements

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Abstract

In this study, we have reconstructed the statistical 3D structure of hundreds of nanometers thick surface immobilized flagellar filament protein layers in their native environment, in buffer solution. The protein deposition onto the surface activated Ta2O5 film was performed in a flow cell, and the immobilization process was followed by in situ spectroscopic ellipsometry. A multilayer optical model was developed, in that the protein layer was described by five effective medium sublayers. Applying this method, an in-depth analysis of the protein layer formation was performed. Based on the kinetics in the distribution of the surface mass density, the statistical properties of the filamentous film could be determined computationally as a function of the measurement time. It was also demonstrated that the 3D structure of the protein layer can be reconstructed based on the calculated in-depth mass density profile. The computational investigation revealed that the filaments can be classified into two individual groups in approximately equal ratio according to their orientation. In the first group the filaments are close to laying position, whereas in the second group they are in a standing position, resulting in a significantly denser sublayer close to the substrate than at a larger distance.Highlights► Flagellar filaments were immobilized onto Ta2O5 substrates in flow-cell. ► The surface was monitored in situ with spectroscopic ellipsometry. ► New optical model was developed for the characterization of the filamentous layers. ► The depth profile of protein mass density was determined. ► The statistical 3D structure of the filamentous layer was reconstructed.

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... These systems are mainly characterized by optical methods [1]. Besides the high sensitivity and sampling speed, optical tools like microscopy, waveguide sensing or ellipsometry are capable of real time monitoring, which is a key to understand the the dynamics of the processes [2][3][4]. Ellipsometry measures phase differences, such as interferometry, however, in the case of ellipsometry the "reference beam" is built in the system in form of the perpendicularly polarized light [5][6][7][8]. This fact is important, because it provides a high sensitivity without the requirement of a coherent source. ...
... Compared to internal reflection configurations (see the next chapter), the conventional (through-liquid) cells have the advantage of measuring on non-transparent substrates, and being sensitive at larger distances from the surface (i.e. for thicker layers). This is important when measuring large molecules, such as flagellar filaments [2][3][4]. The capability of measuring at large distances from the surface allows the reconstruction of long thread-like structures in situ, during adsorption (Fig. 4). ...
... Measurement of the adsorption of flagellar filaments in a regular flow cell[4]. ...
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... Both the instrumentation and the modeling methods are mature. We have shown that complex protein structures can be characterized in situ, using proper optical models [20]. However, in spite of the advanced modeling capabilities, the sensitivity is limited, typically 2-3 orders of magnitude smaller than in case of waveguide sensors. ...
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Protein adsorption is an important aspect for the improvement of many applications, such as medical implants, biosensor design, etc. The density, orientation and conformation of surface-bound proteins are believed to be key factors in controlling subsequent cellular adhesion. The aim of this work is the development of a methodology in order to study in-situ and real-time protein adsorption phenomenon, and describe fibrinogen adsorption on amorphous hydrogenated carbon (a-C:H) thin films developed by rf reactive magnetron sputtering under different deposition conditions. Spectroscopic Ellipsometry (SE) in Vis–UV energy region was implemented for this purpose. SE is a non-destructive, surface sensitive technique, with the capability of performing real-time measurements in air as well as in liquid environment, with great potential in biomedical studies. An appropriate ellipsometric model has been developed, in order to describe accurately the protein adsorption mechanisms in real-time. It was found that the thickness and density of fibrinogen are larger on the a-C:H thin film deposited under absence of bias voltage application. The differences in fibrinogen thickness and transition of fibrinogen from liquid to adsorbed state are presented and discussed in the terms of the surface and optical properties of a-C:H films.
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The thickness resolution and in situ advantage of ellipsometry make this optical technique particularly suitable for studies of thin organic layers of biological interest. Early ellipsometric studies in this area mainly provided thickness quantification, often expressed in terms of surface mass. However, today it is possible to perform monolayer spectroscopy, e.g. of a protein layer at a solid/liquid interface, and also to resolve details in the kinetics of layer formation. Furthermore, complicated microstructures, like porous silicon layers, can be modeled and protein adsorption can be monitored in such layers providing information about pore filling and penetration depths of protein molecules of different size and type. Quantification of adsorption and microstructural parameters of thin organic layers on planar surfaces and in porous layers is of high interest, especially in areas like biomaterials and surface-based biointeraction. Furthermore, by combining ellipsometric readout and biospecificity, possibilities to develop biosensor concepts are emerging. In this report we review the use of ellipsometry in various forms for studies of organic layers with special emphasis on biologically-related issues including in situ monitoring of protein adsorption on planar surfaces and in porous layers, protein monolayer spectroscopy and ellipsometric imaging for determination of thickness distributions. Included is also a discussion about recent developments of biosensor systems and possibilities for in situ monitoring of engineering of multilayer systems based on macromolecules.
Article
A new rank-two variable-metric method is derived using Greenstadt’s variational approach [ Math. Comp. , this issue]. Like the Davidon-Fletcher-Powell (DFP) variable-metric method, the new method preserves the positive-definiteness of the approximating matrix. Together with Greenstadt’s method, the new method gives rise to a one-parameter family of variable-metric methods that includes the DFP and rank-one methods as special cases. It is equivalent to Broyden’s one-parameter family [ Math. Comp. , v. 21, 1967, pp. 368–381]. Choices for the inverse of the weighting matrix in the variational approach are given that lead to the derivation of the DFP and rank-one methods directly.
Article
This paper presents a more detailed analysis of a class of minimization algorithms, which includes as a special case the DFP (Davidon-Fletcher-Powell) method, than has previously appeared. Only quadratic functions are considered but particular attention is paid to the magnitude of successive errors and their dependence upon the initial matrix. On the basis of this a possible explanation of some of the observed characteristics of the class is tentatively suggested.
Article
Bacterial motility involves switching between the left and right supercoiled states of the flagellar filament. The polymorphism of this assembly of identical flagellin molecules has presented a structural puzzle. Supercoiling has been attributed to coexistence of two conformational states of the 11 nearly axially aligned protofilament strands of subunits. The helical parameters of straight filaments in the left (L) and right (R) lattice states have now been accurately determined by X-ray fiber diffraction. The 9 Å resolution electron density map of the R-type filament, refined from the X-ray data, reveals the interlocked -helical segments of the core portion, which constitute the inner and outer tubes. While the inner-tube domain interactions remain invariant, the strand joints in the outer tube can switch between the L- and R-state by 2−3 Å axial shifts, which change the strand periodicity of 50 Å by 0.8 Å. This bi-stable quaternary switching results in supercoiling. Based on the measured helical parameters of the L and R lattices and the switching model, the twist and curvature calculated for the ten possible supercoils are in quantitative accord with observed supercoiled forms of flagellar filaments.
Article
The method of spectroscopic ellipsometry has been applied to study in situ the adsorption of bovine serum albumin (BSA). The porosity and amount of adsorbed BSA were determined by fitting the ellipsometric data to the Bruggeman effective medium approximation model. The presence of intermediate adsorbed layers of polyelectrolytes was found to increase protein adsorption.
Article
The quality of flagellin films in terms of thickness and homogeneity was investigated by spectroscopic ellipsometry. Flagellin films were prepared in three steps: silanization, glutaraldehyde activation and finally the coating with proteins. The process of film preparation was optimized by varying the duration of the silanization and by testing sticking on different substrates including Si wafer covered with different thickness of silicon-oxide and also covered with a thin film of tantallum pentoxide. Spectroscopic ellipsometry was applied to gain in-depth information on the film properties for the optimization of the immobilization. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
Article
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.
Article
The application of ellipsometry of the study of the adsorption behavior of proteins and synthetic macromolecules at the air-water interface has been investigated. It is shown that for macromolecules the amount adsorbed per unit area, Γ, as determined by ellipsometry, only has a well-defined physical meaning if the refractive-index increment remains constant up to high concentrations present in the adsorbed layer. It has been found experimentally that this conditioned is fulfilled for proteins. The ellipsometric Γ values of some protein agree satisfactorily with those obtained by two independent techniques has been used to investigate the adsorption from solution of κ-casein, bovine serum albumin, and polyvinyl alcohol. For bovine serum albumin, Γ reaches a plateau value of 2.9 mg/m2 for concentrations ≥ 0.05 wt%. The thickness of the adsorbed molecules. For κ-casein, Γ steadily increases with increasing centration and multilayers are formed. The technique provides interesting information on conformational changes in adsorbed macromolecules, on the rate of the process, and on the conditions under which these occur.
Article
The modeling of spectroscopic ellipsometry data is reviewed, and is divided into three phases. The first phase involves the calculation of the Fresnel reflection coefficients for a given layer structure; it is shown that the Abelès formalism provides the most flexibility, and can be readily related to the Berreman formalism for calculations involving anisotropic layers. The second phase is to parameterize the optical functions of each individual layer; several models are reviewed, including effective media, the Lorentz oscillator and a recent parameterization of amorphous semiconductors. The final phase involves the fitting of the spectroscopic ellipsometry data to the model, where different figures of merit of the fitting function are discussed. A proper numerical analysis technique requires that the reduced ¢2 be used as the figure of merit, which will result in the proper weighting of data points, and in obtaining meaningful error limits and a measure of the goodness of fit.
Article
The need for repeated measurements in a wide range of biological testing due to statistical variations is well known. In this paper, we discuss a specific example in which the measurement probe is a spectroscopic ellipsometer. Repeatable results are needed in a wide range of applications such as drug testing, immunoassays and other tests for disease, and fundamental biomaterial research. The present paper seeks to help reduce the non-meaningful causes of lack of repeatability by identifying a large number of externally controllable factors. Another goal of this work was to quantify the effects of many of these factors on ellipsometric measurements. By exploiting the sensitivity of spectroscopic ellipsometry to ultrathin layers, improved ways to detect and quantitatively differentiate biological events can be explored. This initial work was motivated from an interest to distinguish one disease from another or discern effects of one drug from another using the high surface sensitivity of spectroscopic ellipsometry. In this paper, we investigate the example biological system of cholera toxin (CT) in an ELISA structure with monosialoganglioside (GM1).
Article
3-Aminopropyl triethoxy silane (APTES) was deposited onto silicon oxide surfaces under various conditions of solvent, heat, and time, and then exposed to different curing environments, including air, heat, and ethanol. The macro- and micromolecular structure of APTES was probed on different levels using different techniques. The thickness of APTES layers was measured by ellipsometry, macromolecular structures were identified using microscopic methods (scanning electron microscopy and atomic force microscopy), and chemical form was investigated using angle-resolved X-ray photoelectron spectroscopy and also reflection infrared spectroscopy of APTES on gold and aluminum oxide surfaces. Coverage equivalent to one monolayer was achieved using very mild reaction and curing conditions (reaction in dry toluene for 15 min at room temperature, curing in air, or 15 min in 200°C oven), whereas thick layers were made by increasing reaction and curing times. APTES initially adsorbed to the surface, and curing was necessary to complete covalent binding between APTES and the surface. Deposition of APTES from water gave thin layers, probably electrostatically bound to silicon.
Article
The adsorption kinetics of three model proteins—human serum albumin, fibrinogen and hemoglobin—has been measured and compared using three different experimental techniques: optical waveguide lightmode spectroscopy (OWLS), ellipsometry (ELM) and quartz crystal microbalance (QCM-D). The studies were complemented by also monitoring the corresponding antibody interactions with the pre-adsorbed protein layer. All measurements were performed with identically prepared titanium oxide coated substrates. All three techniques are suitable to follow in-situ kinetics of protein–surface and protein–antibody interactions, and provide quantitative values of the adsorbed adlayer mass. The results have, however, different physical contents. The optical techniques OWLS and ELM provide in most cases consistent and comparable results, which can be straightforwardly converted to adsorbed protein molar (‘dry’) mass. QCM-D, on the other hand, produces measured values that are generally higher in terms of mass. This, in turn, provides valuable, complementary information in two respects: (i) the mass calculated from the resonance frequency shift includes both protein mass and water that binds or hydrodynamically couples to the protein adlayer; and (ii) analysis of the energy dissipation in the adlayer and its magnitude in relation to the frequency shift (c.f. adsorbed mass) provides insight about the mechanical/structural properties such as viscoelasticity.
Article
The adsorption and competitive adsorption of collagen and bovine serum albumin (BSA) were directly visualized and quantified using atomic force microscopy (AFM) and imaging ellipsometry. Chemically modified silicon surfaces were used as hydrophilic and hydrophobic substrates. The results showed that collagen and BSA in single component solution adsorbed onto a hydrophobic surface two times more than that onto a hydrophilic surface. The competitive adsorption between collagen and BSA showed that serum albumin preferentially adsorbed onto a hydrophobic surface, while collagen on a hydrophilic surface. In the binary solution of BSA (1 mg/ml BSA) and collagen (0.1 mg/ml), nearly 100% of the protein adsorbed onto the hydrophobic surface was BSA, but on the hydrophilic surface only about 6% was BSA. Surface affinity was the main factor controlling the competitive adsorption.
Article
The adsorption of soluble proteins at solid–liquid interfaces plays a fundamental role in both, nature and industrial areas.The formation of organic molecular films below monolayer coverage can be resolved in real-time with ellipsometry, an optical technique based on the principle of change in state of polarization of light on reflection from an optically flat surface.In this communication studies of adsorption kinetics of the protein bovine serum albumin (BSA) on the substrates TiO2, Au and SiO2 are presented.The results show that the kinetics and degree of adsorption of BSA depend on the substrate material.
Article
Nucleic-acid aptamers have attracted intense interest and found wide applications in a range of areas. In this review, we summarize recent advances in the development of aptamer-based biosensors and bioassay methods, most of which have employed electrochemical, optical and mass-sensitive analytical techniques. Aptamers exhibit many advantages as recognition elements in biosensing when compared to traditional antibodies. They are small in size, chemically stable and cost effective. More importantly, aptamers offer remarkable flexibility and convenience in the design of their structures, which has led to novel biosensors that have exhibited high sensitivity and selectivity. Recently, the combination of aptamers with novel nanomaterials has significantly improved the performance of aptamer-based sensors, which we also review in this article. In view of the unprecedented advantages brought by aptamers, we expect aptamer-based biosensors to find broad applications in biomedical diagnostics, environmental monitoring and homeland security.
Article
Quasi-Newton methods accelerate the steepest-descent technique for function minimization by using computational history to generate a sequence of approximations to the inverse of the Hessian matrix. This paper presents a class of approximating matrices as a function of a scalar parameter. The problem of optimal conditioning of these matrices under an appropriate norm as a function of the scalar parameter is investigated. A set of computational results verifies the superiority of the new methods arising from conditioning considerations to known methods.
Article
This paper presents a new minimization algorithm and discusses theoretically some of its properties when applied to quadratic functions. Results of comparative testing for a set of non-quadratic functions are described and reasons for the observed experimental behaviour are suggested.
Article
Label-free biosensors are devices that use biological or chemical receptors to detect analytes (molecules) in a sample. They give detailed information on the selectivity, affinity, and, in many cases, also the binding kinetics and thermodynamics of an interaction. Although they can be powerful tools in the hands of a skilled user, there is often a lack of knowledge of the best methods for using label-free assays to screen for biologically active molecules and accurately and precisely characterize molecular recognition events. This book reviews both established and newer label-free techniques giving both the expert user and the general reader interested in the technologies and applications behind label-free an insight into the field from expert opinion leaders and practitioners of the technologies. Most importantly, chapters contain worked examples from leaders in the field that take the reader through the basics of experimental design, setup, assay development, and data analysis.
Article
An approach to variable metric algorithms has been investigated in which the linear search sub-problem no longer becomes necessary. The property of quadratic termination has been replaced by one of monotonic convergence of the eigenvalues of the approximating matrix to the inverse hessian. A convex class of updating formulae which possess this property has been established, and a strategy has been indicated for choosing a member of the class so as to keep the approximation away from both singularity and unboundedness. A FORTRAN program has been tested extensively with encouraging results.
Article
The use of planar optical waveguides as substrata for label-free, non-invasive monitoring of cells growing on them is demonstrated. Different submicrometre depths (measured from and perpendicular to the substratum surface) can be selected for monitoring. The so-called symmetry waveguide configuration with a low refractive index waveguide support (nanoporous silica with refractive index approximately 1.2) and a polystyrene waveguiding film with a heat-embossed grating coupler is exploited to obtain practically useful differences between the penetration depths of different waveguide modes. Robust data processing techniques are developed to obtain quantitative information about the cell refractive index profile perpendicular to the substratum from the measured effective refractive indices of the modes. In particular, a method is introduced with which cell refractive index variations above and below a predefined and tunable depth can be separated using two modes. The technique can be extended to more modes to gain even more comprehensive information from predefined submicrometre slices of the cell layer. The introduced methods are also suitable for monitoring the kinetics of changes in cell refractive index profiles.
Article
The terminal regions of flagellin from Salmonella typhimurium have been reported to be disordered in solution, whereas the central part of the molecule contains protease-resistant, compact structural units. Here, conformational properties of flagellin and its proteolytic fragments were investigated and compared to characterize the domain organization and secondary structure of flagellin. Deconvolution analysis of the calorimetric melting profiles of flagellin and its fragments suggests that flagellin is composed of three co-operative units or domains. The central part of the molecule, residues 179 to 418, consists of two domains (G1 and G2), whereas the third domain (G3) is discontinuous, constructed from segments 67 to 178 and 419 to 448. Secondary structure prediction and analysis of far-ultraviolet circular dichroic spectra have revealed that G1 and G2 consist predominantly of beta-structure with a little alpha-helical content. G3 contains almost equal amounts of alpha and beta-structure, while in the terminal parts of flagellin the ordered secondary structure seems to be entirely alpha-helical.
Article
Dynamic images of isolated bacterial flagellar filaments undergoing cyclic transformations were recorded by dark-field light microscopy and an ultrasensitive video camera. Flagellar filaments derived from Salmonella SJ25 sometimes stick to a glass surface by short segments near one end. When such a filament, which is a left-handed helix, was subjected to a steady flow of a viscous solution of methylcellulose, its free portion was found to transform cyclically between left-handed (normal) and right-handed (curly or semi-coiled) helical forms. The transformations did not occur simultaneously throughout the whole length of a filament, but occurred at a transition point, which proceeded along the filament. Each transformation process consisted of three phases: initiation, growth and travel. The magnitudes of the mechanical forces, torque and tension, which were generated on a filament by the viscous flow, were obtained by quantitative hydrodynamic analyses. The torque was found responsible for initiating the transformation. The critical magnitude of torque required to induce the normal to semi-coiled transformation was −11 × 10−19 N m and that for the reverse transformation from the semi-coiled to the normal form was 4 × 10−19 N m. Therefore, the filaments showed the characteristics of hysteresis during the cyclic transformation. New types of unstable right-handed helical forms (medium and large) were also induced by mechanical force.
Article
1. INTRODUCTION 2 2. OVERALL STRUCTURE AND SUBSTRUCTURES 5 2.1 Overall structure and components 5 2.2 Bidirectional rotary motor 5 2.3 Drive shaft 8 2.4 Bushing 8 2.5 Universal joint 9 2.6 Helical propeller 9 2.7 Axial junction 10 2.8 Capping structure 11 3. ASSEMBLY PROCESS OF THE FLAGELLUM 11 3.1 Step by step assembly 11 3.2 Flagellum-specific export apparatus and the channel 12 4. UNIQUE CHARACTERISTICS OF THE FLAGELLAR MOTOR DYNAMICS 13 5. STRUCTURAL DESIGN OF FLAGELLIN FOR ASSEMBLY REGULATION AND POLYMORPHISM 14 5.1 Domain organization and terminal disorder of flagellin 15 5.2 The role of terminal disorder in filament formation and polymorphism 17 5.3 Common structural motif for regulation of self-assembly 21 6. STRUCTURAL DESIGN OF FLAGELLAR FILAMENTS FOR POLYMORPHISM 22 6.1 Polymorphic mechanism 23 6.2 Structures of the filaments deduced by electron microscopy 25 6.2.1 Overview of the electron microscopic studies 25 6.2.2 Helical image reconstruction procedure 27 6.2.3 Structural details of the filament 28 6.3 X-ray fibre diffraction studies 32 6.3.1 Overview of the X-ray studies 32 6.3.2 Orientation of liquid crystalline sols and diffraction patterns 33 6.3.3 Equatorial analysis 35 6.3.4 A preliminary map refined at 11 Åresolution 37 6.4 Overall chain folding of the subunit in the filament 38 6.4.1 Mapping out the terminal and central regions 38 6.4.2 The chain folding and role of each domain 42 6.5 Polymorphic nature of flagellar filament 43 6.5.1 Comparison of the L- and R-type 43 6.5.2 New helical symmetry ‘Lt-type’ 46 6.5.3 Direct comparison of the Lt-type lattice to the other two 48 6.5.4 Plausible conformational changes involved in polymorphism 51 7. PERSPECTIVE 55 8. ACKNOWLEDGEMENTS 55 9. REFERENCES 55
Article
Different types of optical biosensor are critically assessed and compared, based on the belief that a comprehensive understanding of their possibilities—and limitations—is needed for their successful exploitation. © 1997 John Wiley & Sons, Ltd.
Article
Bacterial motility involves switching between the left and right supercoiled states of the flagellar filament. The polymorphism of this assembly of identical flagellin molecules has presented a structural puzzle. Supercoiling has been attributed to coexistence of two conformational states of the 11 nearly axially aligned protofilament strands of subunits. The helical parameters of straight filaments in the left (L) and right (R) lattice states have now been accurately determined by X-ray fiber diffraction. The 9 A resolution electron density map of the R-type filament, refined from the X-ray data, reveals the interlocked alpha-helical segments of the core portion, which constitute the inner and outer tubes. While the inner-tube domain interactions remain invariant, the strand joints in the outer tube can switch between the L- and R-state by 2-3 A axial shifts, which change the strand periodicity of approximately 50 A by 0.8 A. This bi-stable quaternary switching results in supercoiling. Based on the measured helical parameters of the L and R lattices and the switching model, the twist and curvature calculated for the ten possible supercoils are in quantitative accord with observed supercoiled forms of flagellar filaments.
Article
Optical biosensors that exploit surface plasmon resonance, waveguides and resonant mirrors have been used widely over the past decade to analyse biomolecular interactions. These sensors allow the determination of the affinity and kinetics of a wide variety of molecular interactions in real time, without the need for a molecular tag or label. Advances in instrumentation and experimental design have led to the increasing application of optical biosensors in many areas of drug discovery, including target identification, ligand fishing, assay development, lead selection, early ADME and manufacturing quality control. This article reviews important advances in optical-biosensor instrumentation and applications, and also highlights some exciting developments, such as highly multiplexed optical-biosensor arrays.
Article
In the development of biosensors, the immobilization of biomolecules at interfaces played a crucial role. The feasibility of using 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (Glu) to modify silicon surface to immobilize covalently protein for immunoassay with the biosensor based on imaging ellipsometry was investigated. The higher density and stability of human IgG layer could be obtained on the silicon surface modified with APTES and Glu than that on the silicon surface modified with dichlorodimethylsilane (DDS). The human IgG molecules immobilized covalently on APTES-Glu surface bound more anti-IgG molecules than that on DDS surface, which indicated that the human IgG molecules could maintain higher binding capability on APTES-Glu surface. Tween 20 was able to block the undesirable adsorption on APTES-Glu surface, and also enhanced the recognition between human IgG and its antibody on both APTES-Glu and DDS surfaces. The combination of this protein covalent immobilization and the biosensor has the potential to be developed into a fast, simple immunoassay technique.
Article
The study of solution-phase interactions between small molecules and immobilized proteins is of intense interest, especially to the pharmaceutical industry. An optical sensing technique, dual polarization interferometry, has been employed for the detailed study of a model protein system, namely, d-biotin interactions with streptavidin immobilized on a solid surface. Changes in thickness and density of an immobilized streptavidin layer as a result of the binding of d-biotin have been directly measured in solution and in real time. The results obtained from this approach are in excellent agreement with X-ray crystallographic data for the structural changes expected in the streptavidin-D-biotin system. The mass changes measured on binding d-biotin also agree closely with anticipated binding capacity values. Determination of the density changes occurring in the protein adlayer provides a means for differentiation between specific and nonspecific interactions.
  • Z H Wang
  • G Jin
Z.H. Wang, G. Jin, Journal of Immunological Methods 285 (2004) 237-243.
  • P Ying
  • Y Yu
  • G Jin
P. Ying, Y. Yu, G. Jin, Z. Tao, Colloids and Surfaces B: Biointerfaces 32 (2003) 1–10.
  • D G Kurt
  • T Bein
D.G. Kurt, T. Bein, Langmuir 9 (1993) 2965-2973.
  • A Sebestyén
  • B Végh
  • A Szekrényes
  • S Kurunczi
  • F Vonderviszt
A. Sebestyén, B. Végh, A. Szekrényes, S. Kurunczi, F. Vonderviszt, Biokémia 30 (2006) 4.