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In situ ellipsometric study of surface immobilization of flagellar filaments

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In situ ellipsometric study of surface immobilization of flagellar filaments

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Abstract

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.

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... The spectral range of in situ bioellipsometry is usually limited either by the transparency of the water [40,41], the optical components or the lack of information of the dispersion of protein in the UV range. The n and k spectra of protein can usually be fitted using a polynomial [40] and an exponential function, respectively. ...
... The spectral range of in situ bioellipsometry is usually limited either by the transparency of the water [40,41], the optical components or the lack of information of the dispersion of protein in the UV range. The n and k spectra of protein can usually be fitted using a polynomial [40] and an exponential function, respectively. However, as the transmission and absorption results in Fig. S1 show, the polynomial and exponential dispersions must be completed with an oscillator model for an accurate description of the features below 280 nm. ...
... However, due to the absorption of Fgn in the UV region, further investigations were needed prior to the optical modelling. For this reason, Fgn adsorption was also monitored in a conventional flow cell (introduced in Ref. [40]) where the light beam travels through the window and the liquid, to be reflected from the surface of SiO 2 on the Si substrate. An appropriate optical model was built to describe the system without the adsorbed protein layer (consisting of a Sellmeier ambient (PBS) with a SiO 2 /Si structure), thus after a 30-min protein adsorption process only one additional layer was needed in the model to describe the optical properties of the formed protein layer. ...
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... Using these techniques a wide range of applications, e.g. high-sensitivity characterization of protein layer formation [11][12][13], cell adhesion [14,15], monitoring of bacteria [14], interfaces [16] and substrate stability [17], has already been reported. Optical waveguide sensors provide high sensitivity, but only within the exponentially decaying region of the evanescent field, of which penetration depth is typically a few hundred nanometers. ...
... In case of the second measurement, the optical model was completed with a subsequent Cauchy model to describe the protein adsorption. The A, B and C parameters of this model were fixed at 1.45, zero and zero, respectively, where A, B and C are the parameters of the Cauchy dispersion formula [11,13,18]. During the evaluation of the in situ measurements, only the thickness of the adsorbed protein layer was fitted. ...
... The dn/dc ratio is the derivative of n with respect to the protein concentration (c) that can be taken from the literature (0.18 cm 3 /g measured at the wavelength of 632.8 nm) [3,26]. The value of n a was fixed at 1.45 [11,13,18]. ...
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Two surface-sensitive label-free optical methods, grating coupled interferometry (GCI) and spectroscopic ellipsometry (SE) were integrated into a single instrument. The new tool combines the high sensitivity of GCI with the spectroscopic capabilities of SE. This approach allows quantification with complex optical models supported by SE and accurate measurements with the evanescent field of GCI. A flow cell was developed to perform combined and simultaneous investigations on the same sensor area in liquid (or gas) environments. The capabilities of the instrument were demonstrated in simple refractometry and protein adsorption experiments.
... Moreover, spectroscopic ellipsometry provides both density and spectral information allowing to explore more complex models for the evaluation of the measured data than typically used biosensor techniques. As a continuation of previous works [13][14][15]29], we present in this study a more detailed and quantitative in situ and in-depth analyzing measurement method for the protein layers of the promising recognition elements of today's investigations in nanotechnology, of the flagellar filaments (FF). ...
... As it was already presented in previous studies in detail [13,15,29], a three-layer optical model can be applied for the characterization of the substrate structure of interest. The thermally oxidized silicon was modeled by single-crystalline Si as the substrate and a SiO 2 layer (the references were taken from the literature). ...
... Consequently, these films were modeled with the same Cauchy layer applied for Ta 2 O 5 ( Fig. 1) [13]. Placing the activated samples into the flow cell the FF material and the buffer ambient were taken into account by individual Cauchy formulas and these with fixed parameters were coupled in one or maximum two subsequent effective medium (EMA) layers [15,29]. ...
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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.
... Real-time ellipsometry has been used for many decades not only in vacuum chambers for solid state processes [4], but also for studying of biointerfaces and chemistry [1]. Although waveguide sensors [5] offer a better sensitivity than bioellipsometry in the conventional configurations, the latter has also been developed toward new, higher-sensitivity spectroscopic configurations, in order to achieve multi-parameter modeling capabilities for the characterization of complex surface structures [6][7][8][9]. ...
... The aim of this study is to investigate different gold layer thicknesses and configurations for the in situ measurement of protein adsorption, and to identify the best conditions and the corresponding parameters, such as the sensitivity and the limit of detection. This will be compared to results by standard flow cell [8] and surface plasmon resonance (SPR) [13] configurations. ...
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... A comprehensive discussion of waveguide sensors is available in the review article of P. Kozma et al [21].The combination of ellipsometry with QCM has also been demonstrated [22]. The typical in situ ellipsometry configuration utilizes a flow cell equipped with glass windows for the input and output light [15,[23][24][25][26][27][28], in which not only the effect of the window and the absorption of the water, but also the fixed angle of incidence puts constraints. The so called Kretschmann configuration couples the light into the substrate using prisms [29][30][31][32][33][34], providing the illumination from the substrate, and measuring at the close proximity of the surface with the evanescent field, similar to OWLS and GCI. ...
... In Fig. 3, it is a monolayer of protein molecules, which was modeled using the Cauchy dispersion (n = A + B/λ 2 + C/λ 4 ) with the parameters of A = 1.45, B = 0.01 and C = 0 [25,26] (using these parameters, the refractive index is 1.47 at the wavelength of 632.8 nm). The thickness of this adsorbing layer was monitored during the measurement. ...
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... There are several methods suitable for immobilizing flagellar nanotubes on various types of surfaces. 48,49 We chose the DSP-based covalent surface chemistry to have a simple but effective method for producing stable surface-filament attachment that can potentially sustain complex media when the sensor is applied in future measurements. The filament layers were characterized using SPR-SE and AFM 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 33 techniques. ...
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... Depending on the optical properties of the surrounding media, ellipsometry has a typical sensitivity of ≈10 −4 in refractive index units (which corresponds to ≈1 ng/mm 2 for surface mass density changes in a silicon substrate), when using a standard flow cell configuration, in which the sample surface is measured through the liquid [20]. A significant increase of the sensitivity can be reached by surface plasmon enhancement [19,21]. ...
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Enzyme immobilization onto silicon substrates has been investigated by five different coupling procedures. The methods included covalent coupling either through a metal link reagent or silane reagents containing pendant amino or epoxide linkers, an entrapment technique using a thin layer of gelatin, or an adsorption technique using poly-l-lysine. These immobilization procedures were evaluated using glucose oxidase and a simple spectrophotometric method employing Fenton’s reagent. Retention of enzyme activity and surface loading were assessed. The immobilization techniques were also evaluated by electron microscopy to characterize the evenness of the surface coatings. All of the covalent coupling procedures led to surface loadings, approaching 1 pmol mm−2; however, the surfaces appeared irregular on a microscopic scale. The poly-l-lysine adsorption technique provided the smoothest surface. With the exception of the entrapment technique, all immobilization procedures provided immobilized enzyme that retained >75% activity after several weeks of storage.
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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
In surface biology there are numerous studies carried out using single wavelength ellipsometry, especially in the area of macromolecular adsorption on solid surfaces. The results obtained contribute significantly to the understanding of the basic mechanisms of adsorption and surface dynamics of organic molecules, especially of proteins. An example of an area of great importance is biomaterials, where ellipsometry is used as a tool in the process of acquiring knowledge about the biological acceptance of new as well as currently used implant materials. In the area of affinity biosensors, ellipsometry has been suggested as a potential readout principle. Ellipsometry is also a tool in emerging technologies, such as surface molecular engineering with the aim to construct molecular superstructures with predesigned biological functions and to interface biology with electronics. However, in most cases when ellipsometry is applied in biology, it has been used for surface mass determination. The potential in using spectroscopic data for resolving microstructural and dynamic information has not been exploited fully. From the above perspective, this report reviews the use of spectroscopic ellipsometry for studies in surface biology and highlights the advantages it offers. Two main themes are developed. The first is spectroscopy on monolayers of macromolecules with emphasis on determination of their dielectric functions and microstructure. A specific example discussed is ferritin adsorption on gold. The results, including dynamics of both the surface mass and layer microstructure, indicate an adsorption model based on a two-state adsorption mechanism. The second theme is ellipsometrically based biosensor systems. The discussion covers aspects of what imaging ellipsometry can provide in this context and is exemplified by results from affinity biosensor and gas sensor systems.
Article
The flagella of Salmonella can adopt a number of distinct helical forms. This article discusses the design of a subunit which packs to give a helical filament. Polymorphism is explained by small changes in the geometry of the subunit.
Article
Well-oriented sols of straight bacterial flagellar filaments have been obtained by preparing reconstituted flagellar filaments with an appropriate length distribution and choosing appropriate solvent conditions. An average filament length of 300 to 500 nm and the use of solvents with very low concentrations of salt has allowed us to prepare highly fluid sols that make flow orientation possible. X-ray fiber diffraction from these sols has shown distinct layer-line reflections to 3.5 A resolution in the meridional direction. Layer-line intensities have been collected by the angular deconvolution method up to 5 A resolution. The possibility of using a magnetic field to further improve the orientation has been explored and a solvent condition that makes flagellar sols sensitive to the magnetic field has been found. General applicability of the method to other systems is also discussed.
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
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
Biosensors exploit the remarkable specificity of biomolecular recognition to provide analytical tools that can measure the presence of a single molecular species in a complex mixture. A new strategy is emerging in the development of biosensor technologies: molecular-engineering techniques are being used to adapt the properties of proteins to simple, generic detector instrumentation, rather than adapting instruments to the unique requirements of a natural molecule.
Article
ELISA provides a highly sensitive procedure for quantitating antigens and antibodies. In that assay, microwells are coated initially with a specific ligand and then saturated with inert molecules to minimize nonspecific background. Coating can be improved by pretreating the microwells with poly-l-lysine (PLL). Proteins and Tween 20 are most often used to block vacant binding sites in enzyme-linked immunosorbent assay (ELISA). In the present study the blocking effects of Tween 20 and bovine serum albumin (BSA) were estimated using an original novel approach. In the assay the magnitude of saturation of the microwells was quantitated by measuring the enzymatic activity of alkaline phosphatase adsorbed to residual vacant sites in the microwell. Tween 20 completely saturated ELISA microwells at concentrations higher than 2 microg/ml. If the microwells were pretreated with PLL, even high concentrations of the detergent did not completely saturate the wells. In contrast, BSA completely saturated both PLL-treated and nontreated microwells at 5 microg/ml. Complementation of Tween 20-induced saturation of PLL-treated microwells was achieved only by addition of BSA at concentration required for BSA alone to reach complete saturation. This approach is applicable for assessing binding to ELISA microwells of any reagent of choice either as a ligand or as a blocking reagent.
Article
Swimming speed (v) and flagellar-bundle rotation rate (f) of Salmonella typhimurium, which has peritrichous flagella, were simultaneously measured by laser dark-field microscopy (LDM). Clear periodic changes in the LDM signals from a rotating bundle indicated in-phase rotation of the flagella in the bundle. A roughly linear relation between v and f was observed, though the data points were widely distributed. The ratio of v to f (v-f ratio), which indicates the propulsive distance during one flagellar rotation, was 0.27 microm (11% of the flagellar pitch) on average. The experimental v-f ratio was twice as large as the calculated one on the assumption that a cell had a single flagellum. A flagellar bundle was considered to propel a cell more efficiently than a single flagellum.
Article
We have measured the time-resolved adsorption kinetics of the mussel adhesive protein (Mefp-1) on a nonpolar, methyl-terminated (thiolated) gold surface, using three independent techniques: quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance, and ellipsometry. The QCM-D and ellipsometry data shows that, after adsorption to saturation of Mefp-1, cross-linking of the protein layer using NaIO4 transforms it from an extended (approximately 20 nm), water-rich, and hydrogel-like state to a much thinner (approximately 5 nm), compact, and less water-rich state. Furthermore, we show how quantitative data about the thickness, shear elastic modulus, and shear viscosity of the protein film can be obtained with the QCM-D technique, even beyond the Sauerbrey regime, if frequency (f) and energy dissipation (D) measurements measured at multiple harmonics are combined with theoretical simulations using a Voight-based viscoelastic model. The modeling result was confirmed by substituting H2O for D2O. As expected, the D2O substitution does not influence the actual adsorption behavior, but resulted in expected differences in the estimated effective density and shear viscosity. These results provide new insight and understanding about the adsorption kinetics and crosslinking behavior of Mefp-1. They also demonstrate how the above three techniques complement each other for biomolecule adsorption studies.
Article
The structure of the adsorbing layers of native and denatured proteins (fibrinogen, gamma-immunoglobulin, albumin, and lysozyme) was studied on hydrophilic TiO(2) and hydrophobic Teflon-AF surfaces using the quartz crystal microbalance with dissipation and optical waveguide lightmode spectroscopy techniques. The density and the refractive index of the adsorbing protein layers could be determined from the complementary information provided by the two in situ instruments. The observed density and refractive index changes during the protein-adsorption process indicated the presence of conformational changes (e.g., partial unfolding) in general, especially upon contact with the hydrophobic surface. The structure of the formed layers was found to depend on the size of the proteins and on the experimental conditions. On the TiO(2) surface smaller proteins formed a denser layer than larger ones and the layer of unfolded proteins was less dense than that adsorbed from the native conformation. The hydrophobic surface induced denaturation and resulted in the formation of thin compact protein films of albumin and lysozyme. A linear correlation was found between the quartz crystal microbalance measured dissipation factor and the total water content of the layer, suggesting the existence of a dissipative process that is related to the solvent molecules present inside the adsorbed protein layer. Our measurements indicated that water and solvent molecules not only influence the 3D structure of proteins in solution but also play a crucial role in their adsorption onto surfaces.
Article
Specific and nonspecific interactions between antibody-modified probes and substrate-immobilized proteins were monitored by atomic force microscopy (AFM). Probes were modified with anti-ovalbumin IgG antibodies immobilized in either an oriented or a random manner. The oriented immobilization of whole IgG was accomplished through the use of Protein A, and random immobilization was carried out with glutaraldehyde. Nonspecific interactions may lead to false detection of antibody-antigen binding events even when the antigen binding sites are properly positioned by an oriented immobilization strategy. Thus, nonionic and zwitterionic surfactants, including Tween 20, Tween 80, Triton X-100, and CHAPS, were evaluated to determine if nonspecific binding events could be reduced without compromising the desired specific antibody-antigen binding. Enzyme-linked immunosorbent assay and surface plasmon resonance assays were also employed to study antibody-antigen binding as a function of immobilization strategy and surfactant concentration. The data from these studies indicate that Protein A can be used to immobilize whole IgG onto AFM probes for force measurement experiments and that a surfactant is useful for improving the selectivity for such measurements.
Article
With the growing number of fatalities resulting from the 100 or so cancer-related diseases, new enabling tools are required to provide extensive molecular profiles of patients to guide the clinician in making viable diagnosis and prognosis. Unfortunately with cancer-related diseases, there is not one molecular marker that can provide sufficient information to assist the clinician in making effective prognoses or even diagnoses. Indeed, large panels of markers must typically be evaluated that cut across several different classes (mutations in certain gene fragments--DNA; over/under-expression of gene activity as monitored by messenger RNAs; the amount of proteins present in serum or circulating tumor cells). The classical biosensor format (dipstick approach for monitoring the presence of a single element) is viewed as a valuable tool in many bioassays, but possesses numerous limitations in cancer due primarily to the single element nature of these sensing platforms. As such, if biosensors are to become valuable tools in the arsenal of the clinician to manage cancer patients, new formats are required. This review seeks to provide an overview of the current thinking on molecular profiling for diagnosis and prognosis of cancers and also, provide insight into the current state-of-the-art in the biosensor field and new strategies that must be considered to bring this important technology into the cancer field.
Article
The kinetics of adsorption of lysozyme and alpha-lactalbumin from aqueous solution on silica and hydrophobized silica has been studied. The initial rate of adsorption of lysozyme at the hydrophilic surface is comparable with the limiting flux. For lysozyme at the hydrophobic surface and alpha-lactalbumin on both surfaces, the rate of adsorption is lower than the limiting flux, but the adsorption proceeds cooperatively, as manifested by an increase in the adsorption rate after the first protein molecules are adsorbed. At the hydrophilic surface, adsorption saturation (reflected in a steady-state value of the adsorbed amount) of both proteins strongly depends on the rate of adsorption, but for the hydrophobic surface no such dependency is observed. It points to structural relaxation ("spreading") of the adsorbed protein molecules, which occurs at the hydrophobic surface faster than at the hydrophilic one. For lysozyme, desorption has been studied as well. It is found that the desorbable fraction decreases after longer residence time of the protein at the interface.
Article
We functionalized Escherichia coli FliC flagellin proteins to form tailored nanotubes binding single types or pairs of ligands, including divalent cations, fluorescent antibodies, or biotin-avidin-linked moieties such as ferritins. The ratio of each tag in bifunctionalized flagella could be toggled extending their sophistication as nanoscaffolds. Tobacco Etch Virus (TEV) protease site-containing FliCs were cleaved by the cognate protease without filament disintegration, potentiating their use as removable nanolithography masks to deposit attached ligands by protease cleavage.
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