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In situ ellipsometric study of surface immobilization of flagellar filaments
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.
... 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. ...
We designed a Bragg mirror structure with an SiO2 top layer to create a resonance in the ultraviolet wavelength range, near the absorption peak position of various proteins. We demonstrate that the wavelength of enhanced sensitivity can be adjusted by proper design of the 1D photonic structure. The possibility to design the wavelength of enhanced sensitivity supports measurements of better selectivity, optimized for the absorption of the target material. Since the width of the resonant peak in the reflectance spectra can be sharper than those of plasmonics, and they can be positioned at more favourable regions of the instrument and material (e.g., in terms of intensity or selectivity), the sensitivity can exceed those of plasmon-enhanced measurements. In this study we demonstrate the main features of the concept at the example of in situ spectroscopic ellipsometry of fibrinogen adsorption in the Kretschmann-Raether configuration. We realized a resonant peak with a full width at half maximum of 3 nm near the wavelength of 280 nm, which coincides with the absorption maximum of fibrinogen. The influence of depolarization and surface roughness on the measurements, and the potential for improving the current experimental detection limit of 45 pg/mm² is also discussed.
... 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]. ...
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]. ...
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. ...
The Kretschmann-Raether geometry is widely used to investigate the properties of various biological samples and their behavior on different substrates ###citearw11a (mostly on gold surface with/without different functionalization). In this configuration the surface plasmon polaritons (SPPs) are used to enhance the sensitivity of the measurement. Recently, the combination of this method with spectroscopic ellipsometry (SE) became more and more popular. In our work protein adsorption was monitored in situ using this configuration. The performance of the configuration was investigated for different thicknesses of the plasmonic layer. The best measurement parameters were identified in terms of layer thickness, angle of incidence (AOI) and wavelength range. It was shown that the spectroscopic capability over a broad wavelength range, the possibility to adjust the AOI accurately, as well as the phase information from the measurement proves to be a significant advantage compared to standard configuration and surface plasmon resonance configurations.
... 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. ...
Plasmon-enhanced in situ spectroscopic ellipsometry was realized using the Kretschmann geometry. A 10-μL flow cell was designed for multi-channel measurements using a semi-cylindrical lens. Dual-channel monitoring of the layer formation of different organic structures has been demonstrated on titania nanoparticle thin films supported by gold. Complex modeling capabilities as well as a sensitivity of ~40 pg/mm² with a time resolution of 1 s was achieved. The surface adsorption was enhanced by the titania nanoparticles due to the larger specific surface and nanoroughness, which is consistent with our previous results on titanate nanotubes.
... 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. ...
Environmental monitoring of Ni is needed around the WHO threshold limit of 0.34 µM. This sensitivity target can usually only be met by time consuming and expensive laboratory measurements. There is a need for cheap field-applicable methods, even if it is only used for signaling the necessity of a more accurate laboratory investigation. In this work bio-engineered protein-based sensing layers were developed for Ni detection in water. The bacterial Ni-binding flagellin variants were fabricated using genetic engineering, and their applicability as Ni-sensitive biochip coatings was tested. Nanotubes of mutant flagellins were built by in vitro polymerization. A large surface density of the nanotubes on the sensor surface was achieved by covalent immobilization chemistry based on a dithiobis(succimidyl propionate) crosslinking method. The formation and density of the sensing layer was monitored and verified by spectroscopic ellipsometry and atomic force microscopy. Cyclic Voltammetry (CV) measurements revealed a Ni sensitivity below 1 µM. It was also shown that even after two months storage the used sensors can be regenerated and re-used by rinsing in 10 mM solution of ethylenediaminetetraacetic acid at room temperature.
... 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]. ...
Optical methods have been used for the sensitive characterization of surfaces and thin films for more than a century. The first ellipsometric measurement was conducted on metal surfaces by Paul Drude in 1889. The word 'ellipsometer' was first used by Rothen in a study of antigen-antibody interactions on polished metal surfaces in 1945. The 'bible' of ellipsometry has been published in the second half of the '70s. The publications in the topic of ellipsometry started to increase rapidly by the end of the '80s, together with concepts like surface plasmon resonance, later new topics like photonic crystals emerged. These techniques find applications in many fields, including sensorics or photovoltaics. In optical sensorics, the highest sensitivities were achieved by waveguide interferometry and plasmon resonance configurations. The instrumentation of ellipsometry is also being developed intensively towards higher sensitivity and performance by combinations with plasmonics, scatterometry, imaging or waveguide methods, utilizing the high sensitivity, high speed, non-destructive nature and mapping capabilities. Not only the instrumentation but also the methods of evaluation show a significant development, which leads to the characterization of structures with increasing complexity, including photonic, porous or metal surfaces. This article discusses a selection of interesting applications of photonics in the Centre for Energy Research of the Hungarian Academy of Sciences.
... In this work, we discuss the behavior of the PI in aqueous solutions, and we present our investigations of the PEI deposition on PI surface, and its stability. Our main measurement technique was in situ spectroscopic ellipsometry, which ensures many possibilities to study thin layers [9,10]. As the measurements immediately revealed a large drift of the measured signal for PI in aqueous solutions, the protection of the PI layer was also aimed. ...
Novel biosensors made of polymers may offer advantages over conventional
technology such as possibility of mass production and tunability of the
material properties. With the ongoing work on the polymer photonic chip
fabrication in our project, simple model samples were tested parallel
for future immobilization and accessing conditions for applications in
typical aqueous buffers. The model samples consist of a thin, high
refractive index polyimide film on top of TEOS on Si wafer. These model
samples were measured by in situ spectroscopic ellipsometry using
different aqueous buffers. The experiments revealed a high drift in
aqueous solutions; the drift in the ellipsometric parameters (delta,
psi) can be evaluated and presented as changes in thickness and
refractive index of the polyimide layer. The first molecular layer of
immobilization is based on polyethyleneimine (PEI). The signal for the
PEI adsorption was detected on a stable baseline, only after a long
conditioning. The stability of polyimide films in aqueous buffer
solutions should be improved toward the real biosensor application.
Preliminary results are shown on the possibilities to protect the
polyimide. Optical Waveguide Lightmode Spectroscopy (OWLS) has been used
to demonstrate the shielding effect of the thin TiO2 adlayer
in biosensor applications.
Electrochemical migration is a critical factor contributing to failures in electronics due to humidity. When moisture accumulates on conductor-dielectric-conductor systems under bias voltage, electrochemical processes can be triggered, leading to the growth of metallic dendrites that may ultimately result in system failure. Despite its significance, many aspects of electrochemical migration remain unresolved, particularly regarding the physical characteristics of liquid buildup that facilitate dendrite growth and short circuit currents. While there are a few techniques that can measure water adsorption on the nanoscale, most conventional methods focus on water droplets within the size range of visible light wavelengths. In this study, we implemented a combined electrical-optical-ellipsometric measurement on FR-4 printed circuit boards featuring Sn surface finishes. Our experimental setup allowed for the measurement of water condensation across a wide range of thicknesses, while simultaneously monitoring the solder mask and metal electrodes during cooling. The ambient temperature of 25 ∘C and a relative humidity of 60% were constant during the measurement. By employing this approach, we elucidated the mechanisms of dendrite formation and short circuit currents, demonstrating that the water film remains continuous between droplets on the solder mask surface. Compared to Sn the nucleation was delayed on the solder mask with a larger surface coverage at smaller thicknesses. This comprehensive methodology provides crucial insights into the electrochemical migration process, enhancing our understanding of the underlying phenomena that contribute to electronic failures due to humidity. Our work highlighted the complementary nature of ellipsometry and optical imaging.
Understanding interface processes has been gaining crucial importance in many applications of biology, chemistry, and physics. The boundaries of those disciplines had been quickly vanishing in the last decade, as metrologies and the knowledge gained based on their use improved and increased rapidly. Optical techniques such as microscopy, waveguide sensing, or ellipsometry are significant and widely used means of studying solid‐liquid interfaces because the applicability of ions, electrons, or X‐ray radiation is strongly limited for this purpose due to the high absorption in aqueous ambient. Light does not only provide access to the interface making the measurement possible, but utilizing the phase information and the large amount of spectroscopic data, the ellipsometric characterization is also highly sensitive and robust. This article focuses on ellipsometry of biomaterials in the visible wavelength range. The authors discuss the main challenges of measuring thickness and optical properties of ultra‐thin films such as biomolecules. The authors give an overview on different kinds of flow cells from conventional through internal reflection to combined methods. They emphasize that surface nanostructures and evaluation strategies are also crucial parts of in situ bioellipsometry and summarize some of the recent trends showing examples mainly from their research.
Indirect optical methods like ellipsometry or scatterometry require an optical model to calculate the response of the system, and to fit the parameters in order to minimize the difference between the calculated and measured values. The most common problem of optical modeling is that the measured structures and materials turn out to be more complex in reality than the simplified optical models used as first attempts to fit the measurement. The complexity of the optical models can be increased by introducing additional parameters, if they (1) are physically relevant, (2) improve the fit quality, (3) don't correlate with other parameters. The sensitivity of the parameters can be determined by mathematical analysis, but the accuracy has to be validated by reference methods. In this work some modeling and verification aspects of ellipsometry and optical scatterometry will be discussed and shown for a range of materials (semiconductors, dielectrics, composite materials), structures (damage and porosity profiles, gratings and other photonic structures, surface roughness) and cross-checking methods (atomic force microscopy, electron microscopy, x-ray diffraction, ion beam analysis). The high-sensitivity, high-throughput, in situ or in line capabilities of the optical methods will be demonstrated by different applications.
For several decades, biosensors are becoming increasingly developing through their areas of application. In this context, through a multidisciplinary approach, we designed a measuring cell and microfluidic chips of PDMS polymer, coupled with Love wave immunosensor, to provide a platform for the rapid detection of bio-organisms. This new platform allowed direct and specific antibodies and Escherichia Coli whole living bacteria detection. It also helped to significantly reduce the detection time (from several hours to a few minutes), simplify testing protocols, and introduce "dynamic behaviour" of biological phenomena. The specificity of the detection is based on the functionalization of the sensor surface, the platform can be adapted to the detection of a wide spectrum of bioorganisms compounds or in a liquid medium.
The absorption spectra of different aqueous dispersions containing silver nanoparticles were computed by finite element method and compared to spectra determined by UV-Visible spectroscopy. This comparative study proved that the spectrum measured on the aqueous dispersion of bare silver nanoparticles with absorptance maximum at lambdameas = 391 nm corresponds to the characteristic UV surface plasmon band of spherical nanoparticles with 8.25 nm diameter. The presence of aggregates in aqueous dispersions resulted in splitting on the spectrum, when the surface of the silver nanoparticles with cAg = 2×10-4 M concentration was functionalized by L-cysteine with cCys=5.7×10-6 M concentration. The simplest aggregate-geometries that exhibit resonance at the measured absorptance maxima were determined by varying the number of silver nanoparticles, and the inter-particle distance in linear chains, and taking 0.45 nm thick cysteine-shell into account. The FEM computations proved that the primary maxima in the UV involve quadrupolar modes, while the secondary maxima red-shifted to lambdameas_2=567 nm at pH=2.98 and lambdameas_2'=588 nm at pH=4.92 originate from coupled dipolar plasmon resonances on extended aggregates aligned along the E-field oscillation direction. The non-aggregated particles and the aggregates rotated with respect to the E-field oscillation direction significantly contribute to the UV peak.
Citrate-stabilized spherical silver nanoparticles (Ag NPs) with d=8.25±1.25 nm diameter were prepared and functionalized with L-cysteine (Cys) in aqueous dispersion. The nanosilver-cysteine interactions have been investigated by Raman and (1)H NMR spectroscopy. The effect of pH on stability of biofunctionalized Ag NPs was investigated. The cysteine-capped nanosilver dispersions remain stable at higher pH (pH>7), while the degree of aggregation increased as the pH decreased. Below pH ~7, the characteristic surface plasmon band of bare silver nanoparticles was back-shifted from λ(measured)(bareAgNP)=391 nm to λ(measured)(1)=387-391 nm, while the presence of a new band at λ(measured)(2)=550-600 nm was also observed depending on pH. Finite element method (FEM) was applied to numerically compute the absorption spectra of aqueous dispersions containing bare and cysteine-functionalized Ag NPs at different pH. Both the dynamic light scattering (DLS) measurements, Zeta potential values and the transmission electron microscopic (TEM) images confirmed our supposition. Namely, electrostatic interaction arose between the deprotonated carboxylate (COO(-)) and protonated amino groups (NH(3)(+)) of the amino acid resulting in cross-linking network of the Ag NPs between pH ~3 and 7. If the pH is measurable lower than ~3, parallel with the protonation of citrate and L-cysteine molecules the connection of the particles via l-cysteine is partly decomposed resulting in decrease of second plasmon band intensity.
Here we lay the ground work for the detection of hepatitis C viral NS3-4A protease exploiting peptide-protein interaction. The NS3-4A protease is inhibited by N-terminal cleavage products. Our approach is based on the formation of a self-assembled monolayer (SAM) of a ferrocene amino acid derivative on an electrode surface. A short NS3-4A specific inhibitory peptide (Asp-Glu-Ile-Val-Pro-Nva) was then covalently attached to the electrode surface. The interaction of the peptide, through the C-terminal, with the protein was quantified using electrochemical techniques. The systems exhibit a linear relationship between the measured signal and NS3-4A concentration in the range of 10-100 pM with a detection limit of 5 pM.
Cavities created by He implantation with a dose of 5×1016 cm−2 and energy of 40 keV into single-crystalline silicon and annealing at 650–1000 °C for 15–60 min were characterized by multiple angles of incidence spectroscopic ellipsometry. Optical models of increasing complexity were developed assuming the cavity layer either to be homogeneous, or to have a Gaussian profile, or sublayers with independently fitted cavity ratios. Cavity profiles of different annealing conditions were compared and cross-checked by transmission electron microscopy. A strategy for the ellipsometric evaluation was proposed to reduce the computation time and the probability of getting in local minima using complex models with numerous parameters. High sensitivity on the angle of incidence was found, and the choice and the determination of the angle of incidence were discussed.
An expression system for studying epitopes of adhesion proteins based on fusion of gene fragments into fliC(H7) of Escherichia coli is described. We constructed the system by an in-frame insertion of DNA fragments encoding one, two or three of the fibronectin-binding D repeats present in the fibronectin-binding protein A (FnBPA) of Staphylococcus aureus, into the fliC(H7) gene region encoding the variable domain of the H7 flagellin. The constructs were expressed by in trans complementation in the E. coli strain JT1 which harbours knock-out mutations for the expression of FliC as well as of the mannoside-binding fimbrial adhesin. The resulting chimeric flagella, which contained 39, 77 or 115 heterologous amino acid residues, efficiently bound soluble and immobilized human plasma and cellular fibronectin, and the binding was most efficient with the flagella containing the three D repeats of FnBPA. The chimeric flagella bound to frozen sections of human kidney and to cultured human cells. Antibodies raised against the chimeric flagella bound to Protein A-deficient S. aureus cells and inhibited the binding of staphylococci to immobilized fibronectin. We also expressed peptides, ranging in size between 48 and 302 amino acids, of the collagen-binding YadA adhesin of Yersinia enterocolitica. A fragment of 302 amino acids representing the middle region of YadA was needed for collagen binding. Chimeric flagellar filaments expressing hundreds of intimately associated adhesive epitopes offer versatile tools to analyze adhesin-receptor interactions and functional epitopes of adhesion proteins.
Optical waveguide lightmode spectroscopy has been used to observe the deposition of bacterial flagellar filaments of mean length 350 nm from bulk solution onto a smooth planar substratum, chemically modified to covalently bind the flagellar filaments on contact. At the highest practicable bulk concentration, the filaments follow the theoretically predicted kinetics of random sequential addition of highly elongated rigid rods to the substratum, but addition terminates with the rods almost perpendicular to the substratum. Rod-rod correlations in the bulk anomalously accelerate the rate of arrival of the filaments at the surface of the substratum, relative to spheres. At lower concentrations, this effect is absent, and the rods have time to order themselves on the substratum, forming a two-dimensional array.
A novel organosilane coupling agent is disclosed and its use as an adhesion promoter in mineral-filled unsaturated polymer systems is described. Additionally, use of the organosilane as a primer for various substrates is presented. The coupling agent comprises the reaction product of an isocyanatoalkyl ester of acrylic or methacrylic acid with an aminoorganosilane. The organosilane so formed links the acryloxyalkyl or methacryloxyalkyl functionality to an alkylene, or aminoalkylene, group on the silicon atom through a urea group. Use of the organosilane as a coupling agent in a mineral-filled unsaturated polymer results in superior resistance to moisture, particularly when the polymer is selected from the group of corrosion resistant unsaturated polyesters.
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.
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.
Anisotropic intermolecular potentials for Ar · HF, Kr · HF and Xe · HF are obtained by least squares fitting to molecular beam spectra of van der Waals complexes. The absolute minimum is at the linear rare gas-HF geometry for all the systems considered. The potentials are reliable around the absolute minimum, but the existing data are not sensitive to the behaviour elsewhere. In particular, the absolute well depths and the behaviour around the linear rare gas-FH geometries are not well determined and depend upon the model chosen. Experiments which would provide further information on the potentials for HF systems are suggested.The potentials for the rare gas-HF systems are considerably more anisotropic than for the corresponding rare gas-HCl systems. The contributions of induction and dispersion forces to the potential anisotropy are discussed and both are found to be significant. It seems likely that the attractive forces can be adequately described in terms of induction and dispersion forces alone, without the need to invoke incipient chemical bonding.
The adsorption of some model proteins, human serum albumin (HSA), IgG, fibrinogen, and lysozyme, at methylated silica surfaces was investigated with in situ ellipsometry. By performing studies with the bare substrate at two different ambient refractive indices and using four-zone averaging, the adsorbed amount, the adsorbed layer thickness, and the mean adsorbed layer refractive index are obtained. The adsorbed amounts obtained for the proteins agree well with previous results. The adsorbed layer thicknesses vary strongly between the proteins. Thus, at adsorption plateau, the adsorbed layer thicknesses (δel) obtained for HSA, lysozyme, IgG, and fibrinogen are 4 ± 2, 11 ± 2, 18 ± 2, and 28 ± 2 nm, respectively. The buildup of the adsorbed layers proceeds differently for different proteins. Thus, for fibrinogen, both δel and the adsorbed layer mean refractive index (nf) increase monotonically up to about 4 mg/m2. For IgG, on the other hand, δel is essentially independent of the adsorbed amount, whereas nf increases linearly. Finally, the adsorbed layer formed by lysozyme is more compact than those formed by fibrinogen, IgG, and HSA. These findings are discussed in terms of adsorbed layer structure.
Biosensors are a cunning combination of biological molecules and microelectronics that can be used to measure blood glucose
levels, pollutants in the environment or food-borne pathogens in the food supply. In a comprehensive TechView, Anthony Turner
takes us on a tour of historical developments and the latest innovations in biosensor research.
The previously described FliTrx E. coli flagellin protein was genetically engineered to display rationally designed histidine, arginine-lysine, and aspartic acid-glutamic acid peptide loops on the solvent-accessible outer domain region. The resulting flagellin monomers were self-assembled to obtain the corresponding oligomeric flagella bionanotubes in which the peptide loops were 5 nm apart. These flagella nanotubes were equilibrated with solutions of various metal ions (Co(II), Cu(II), Cd(II), Ag(I), Au(I), and Pd(II)). Controlled reduction of these metal ions yielded ordered arrays of nanoparticles or nanotubes, and in some cases, extensive aggregation resulted in formation of metal nanotube bundles. Both metal nanoparticles and nanotubes were generated with Cu(II) and Au(I), depending on the initial concentration of Cu(II) ions, while Ag(I) consistently formed metal nanowires, even under relatively mild conditions of reduction. The covalent attachment of separately synthesized Au nanoparticles to the flagella scaffold was also demonstrated. Controlled reduction of Co(II), Cd(II), and Pd(II) complexed with histidine and aspartic acid-glutamic acid peptide loops yielded ordered arrays of the respective metal nanoparticles on individual flagella nanotubes with minimal aggregation. The peptide loop modified flagella nanotubes have been demonstrated to be useful scaffolds for the generation of ordered arrays of metal nanoparticles or uniform nanotubes.
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)
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.
The refractive index increment of a protein solution is a property not only of the protein, but also of the solvent. This is demonstrated theoretically and confirmed experimentally using analytical interferometry. © 1998 John Wiley & Sons, Inc. Biopoly 46: 489–492, 1998
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 μm (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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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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