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Plasmon-enhanced two-channel in situ Kretschmann ellipsometry of protein adsorption, cellular adhesion and polyelectrolyte deposition on titania nanostructures

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Abstract

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.

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... Filaments with Ni-binding 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 8 ability were deposited by thiol-based surface chemistry as a thin layer on the working electrode of an electrochemical microsensor specially designed for this purpose. The immobilization of the developed metal-binding filaments was followed by a recently developed in situ optical method 30,31 and the prepared filament layers were also characterized using atomic force microscopy (AFM). To assess the sensitivity, detection limit, reproducibility and regenerability, the deposited filament layers were electrochemically tested in buffer solution (pH 7.4) by CV. ...
... 37 By utilizing its high sensitivity, SE has been long used as an excellent tool for following bio-related processes. [38][39][40] In this study, a Woollam M2000 rotating compensator spectroscopic ellipsometer was used with a home-made Kretschmann-Raether flow cell ( Fig. 2) for internal reflection surface plasmon-enhanced ellipsometry (SPR-SE) 30,31 that utilizes the surface plasmon resonance (SPR) effect. 41 SPR is one of the most sensitive optical methods in the field of bio-related research, and its combination with SE 15 provides further advantages, such as the possibility of the construction of a complex optical model and the utilization of the phase information. ...
... This method is called two-channel spectroscopic ellipsometry ("channel" refers here to the measurement channels). 30 The SPR-SE measurement was carried out in a wide wavelength range of 400-1690 nm and at the optimal angle of incidence that had been carefully chosen prior to the adsorption process. The angle of incidence was held constant during the measurement. ...
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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.
... (ii) The angle of incidence can be changed arbitrarily, although the very large and very small angles are usually limited by geometrical reasons, and using special arrangement the multiple-angle capabil- ity can also be realized in the case of conventional flow cells [66]. (iii) The sensitivity can be further enhanced by special surface layers and structures [67][68][69][70] (being especially sensitive to the z direction). (iv) Small cell volumes can easily be realized. ...
... Consequently, this method is a multichannel measurement only from the point of view of the detection, the flow channel is the same for the different locations. This way, it can be ensured that all the measurement and analyte parameters are the same, only the surfaces are different, which helps us to get rid of the systematic errors [70]. ...
... In the case of using Bragg multilayers the width and position of the resonance, the optimum angle of incidence and many other parameters can be tuned by changing the materials, numbers and thicknesses of the layers, which results in a much larger freedom in design than in the case of plasmonic layers. 5 Nanostructured surfaces Surface nanostructures have primarily been used to enhance the adsorption of biomolecules [70], however, they can also be used to enhance the sensitivity by increasing the change in the optical signal that was generated by the same amount of protein. ...
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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.
... In this configuration the interface is reached from the substrate, and measured by the evanescent field at the proximity of the interface. Using this concept, illuminating the interface through a semi-cylinder (figure 1), a wide range of incident angles can be used with a standard spectroscopic ellipsometer [22]. The performance of this setup has been demonstrated for different plasmonic gold layer thicknesses [23]. ...
... The focusing allows the use of a small volume (10 µL) flow cell with a channel length of 8 mm. When scanning the spot along the long axis of the channel that coincides with the axis of the cylinder, multi-surface (multichannel) measurements are possible within one experiment [22]. The internal reflection, plasmonenhanced measurement can also be combined with imaging ellipsometry [26]. ...
... A different method Figure 1. Schematic drawing of a 10-µL Kretschmann-Raether flow cell (left, a , m and 0 denote the dielectric fucntions of the BK7 glass semi-cylinder, the gold layer and the water in the flow cell, respectively; θ a is the angle of incidence, d stands for the thickness of the gold layer), as well as the realized construction that can readily be put on the mapping stage of the ellipsometer (middle, see also Ref. [22]). To the right, the schematic view of the dual-channel configuration is shown. ...
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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.
... The investigation of the adsorption of biomolecules and objects of similar size is of primary importance in biology and many other fields. [1] Spectroscopic ellipsometry (SE) is a sensitive and nondestructive method [2] that can monitor surface processes [3,4] with high temporal resolution (one spectroscopic measurement can take a fraction of a second). There are many sensitive optical methods for effective solid-liquid interface characterizations that are mainly based on waveguide sensing, [5] however, the reliable quantification, interpretation and complex modeling of the measured structures remains a challenge, especially for highly sensitive plasmonic configurations. ...
... There are many sensitive optical methods for effective solid-liquid interface characterizations that are mainly based on waveguide sensing, [5] however, the reliable quantification, interpretation and complex modeling of the measured structures remains a challenge, especially for highly sensitive plasmonic configurations. [3] Among other groups we also work on the combination of those methods with ellipsometry, towards the aim of and advanced modeling. [6] Like in most metrologies, quantification is a great challenge, [7,8] which can be handled by comparative metrology based on reference methods. ...
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Bovine fibrinogen monolayers on thin gold films and glassy carbon substrate were investigated using grazing incidence X-ray fluorescence (GIXRF) and spectroscopic ellipsometry (SE). The aim was to determine the amount of protein and to develop models and references for the SE measurement. Both methods were capable of measuring protein amount in the range of μg cm−2 with a sensitivity below 10%, which suggests the use of both techniques as complementary, combined methods. To do it with a high confidence, the lateral uniformity and the stability of the layers during transportation has to be investigated in more detail in the future.
... Another interesting venture is the combination of GCI with spectroscopic ellipsometry (GCI-SE) in one instrument, which may offer the simultaneous exploitation of the high sensitivity provided by GCI and the spectroscopic capabilities of SE, allowing complex multilayer structures to be analyzed in detail [278]. SE-SPR is a (i) type combination realizing the generation of surface plasmons and simultaneous SE measurement in the proper configuration [279][280][281][282][283]. Whether the data are obtained from (i) or (ii), the postmeasurement combination of QCM and optical data are present complex data analysis challenges. ...
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... Another interesting venture is the combination of GCI with spectroscopic ellipsometry (GCI-SE) in one instrument, which may offer the simultaneous exploitation of the high sensitivity provided by GCI and the spectroscopic capabilities of SE, allowing complex multilayer structures to be analyzed in detail [278]. SE-SPR is a (i) type combination realizing the generation of surface plasmons and simultaneous SE measurement in the proper configuration [279][280][281][282][283]. Whether the data are obtained from (i) or (ii), the postmeasurement combination of QCM and optical data are present complex data analysis challenges. ...
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Interfacial layers are important in a wide range of applications in biomedicine, biosensing, analytical chemistry and the maritime industries. Along with the growing number of applications, the analysis of these layer properties and understanding their behavior is becoming crucial. Label-free surface sensitive methods are excellent tools to monitor the structure and formation kinetics of thin layers in real-time even at the nanoscale. In this study, we review the available label-free techniques and demonstrate how the experimentally obtained data can be utilized to extract kinetic and structural information about the formed layers. We outline the most traditional and some novel techniques using optical and mechanical transduction principles. With a special focus, we review the current possibilities o the combination of label-free methods which is a powerful tool to extend the range of detected parameters. We summarize the most important theoretical considerations for obtaining reliable information from measurements taking place in liquid environments and hence layers in a hydrated state. Detailing with both structural and kinetic analysis methods, a thorough overview of the various quantities obtained from the evaluation of the raw label-free data are also provided. Here, thickness, refractive index, optical anisotropy and the related chain orientation, hydration degree, viscoelasticity, footprint, association and dissociation rate constants are all mentioned in detail. To demonstrate the effect of variations in model conditions on the observed data, simulations of kinetic curves at various model settings are also included. Based on our own experience with optical waveguide lightmode spectroscopy (OWLS) and the quartz crystal microbalance (QCM), we have developed software packages for data analysis. Along with this paper, we make the developed software available for the wider scientific community.
... We also utilized an improved hemisphere for the KR ellipsometry (KRSE) setup that contributed to the outstanding signal-tonoise ratio in the crucial spectral range below 300 nm. The KR cell can be mounted on the mapping stage of the ellipsometer [32], and the optical adjustment of the system is supported by the control of the mapping stage. The optical parameters of the KR setup (focusing lenses, hemisphere, glass slide, index matching liquid) enabled us to use the = 200-1690 nm spectral range of the ellipsometer. ...
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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.
... So far, the most successful tool to tackle the challenge of exploring the real-time kinetics of cell adhesion has been the application of surface sensitive label-free methods (surface plasmon resonance (SPR) 29,30 , infrared SPR [31][32][33] , photonic crystals [34][35][36] , quartz crystal microbalance [37][38][39] , ellipsometry 40,41 , digital holographic microscopy 42,43 ), most of them are using surface bound evanescent waves. For example, the resonant waveguide grating (RWG) based optical biosensors measure the local refractive index change in the 150 nm vicinity of the sensor surface, thus providing real-time kinetic information on cell adhesion and spreading, typically on a population of cells (Fig. 1a). ...
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Single-cell adhesion force plays a crucial role in biological sciences, however its in-depth investigation is hindered by the extremely low throughput and the lack of temporal resolution of present techniques. While atomic force microcopy (AFM) based methods are capable of directly measuring the detachment force values between individual cells and a substrate, their throughput is limited to few cells per day, and cannot provide the kinetic evaluation of the adhesion force over the timescale of several hours. In this study a high spatial and temporal resolution resonant waveguide grating based label-free optical biosensor was combined with robotic fluidic force microscopy to monitor the adhesion of living cancer cells. In contrast to traditional fluidic force microscopy methods with a manipulation range in the order of 300–400 micrometers, the robotic device employed here can address single cells over mm-cm scale areas. This feature significantly increased measurement throughput, and opened the way to combine the technology with the employed microplate-based, large area biosensor. After calibrating the biosensor signals with the direct force measuring technology on 30 individual cells, the kinetic evaluation of the adhesion force and energy of large cell populations was performed for the first time. We concluded that the distribution of the single-cell adhesion force and energy can be fitted by log-normal functions as cells are spreading on the surface and revealed the dynamic changes in these distributions. The present methodology opens the way for the quantitative assessment of the kinetics of single-cell adhesion force and energy with an unprecedented throughput and time resolution, in a completely non-invasive manner.
... Â is the angle of incidence and d is the thickness of the gold layer. configuration ( Fig. 1) [10,11], in which the interface is measured from the substrate [2] through a plasmonic gold layer [12]. This configuration can utilize a larger range of both wavelengths and angles of incidence, as well as an additional plasmon enhancement. ...
Article
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.
... This challenge led to an intense research to study adhesion of polypeptides onto silicon and other semiconductor surfaces. Spectroscopic ellipsometry is an appropriate tool for the experimental investigation of biofilms and biological materials because of its non-invasive manner and outstanding sensitivity, which enables to follow the adsorption properties of large organic molecules[1][2][3][4][5][6]. During the evaluation of the ellipsometric measurements, the structure and the optical functions of the bio-layer have to be modeled, which process can be difficult (e.g. ...
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Integrated planar optical waveguide interferometer biosensors are advantageous combinations of evanescent field sensing and optical phase difference measurement methods. By probing the near surface region of a sensor area with the evanescent field, any change of the refractive index of the probed volume induces a phase shift of the guided mode compared to a reference field typically of a mode propagating through the reference arm of the same waveguide structure. The interfering fields of these modes produce an interference signal detected at the sensor׳s output, whose alteration is proportional to the refractive index change. This signal can be recorded, processed and related to e.g. the concentration of an analyte in the solution of interest. Although this sensing principle is relatively simple, studies about integrated planar optical waveguide interferometer biosensors can mostly be found in the literature covering the past twenty years. During these two decades, several members of this sensor family have been introduced, which have remarkably advantageous properties. These entail label-free and non-destructive detection, outstandingly good sensitivity and detection limit, cost-effective and simple production, ability of multiplexing and miniaturization. Furthermore, these properties lead to low reagent consumption, short analysis time and open prospects for point-of-care applications. The present review collects the most relevant developments of the past twenty years categorizing them into two main groups, such as common- and double path waveguide interferometers. In addition, it tries to maintain the historical order as it is possible and it compares the diverse sensor designs in order to reveal not only the development of this field in time, but to contrast the advantages and disadvantages of the different approaches and sensor families, as well.
Article
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.
Article
A phase shifting optical waveguide interferometer is demonstrated for label-free evanescent wave biosensing in aqueous solutions. In the proposed configuration, the reference and measurement arms of the interferometer are combined inside an integrated optical Ta2O5 waveguide using an ion implanted grating. Biomolecules adsorbing on the waveguide surface shift the phase in the measurement arm, which is modulated at the same time by a periodically relaxing liquid crystal phase modulator. It is demonstrated that by analyzing the periodic intensity response at the end facet of the waveguide, the phase shifts in the measurement arm can be monitored in real-time with a precision of 10−4rad. The high phase resolution allows the detection of surface adsorbed molecule densities below 1pg/mm2 without using any labeling or on-chip referencing. The instrument performance is demonstrated by monitoring in situ protein adsorption and affinity binding of low molecular weight pure biotin to an immobilized avidin layer.
Article
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.
Article
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
We have used spectroscopic ellipsometry to determine the dielectric functions of thin films of γ-globulin, bovine serum albumin, and hemoglobin in the visible and near-uv photon energy range. We show that both thickness and dielectric function can be resolved for monomolecular films adsorbed on substrates with relatively low polarizability. The data which we consider to be closest to the intrinsic dielectric response of the protein films were obtained on HgTe and HgCdTe substrates. Less resolution was obtained on silicon substrates. For a density-deficient film, we were able to model the dielectric response with effective medium theories, and the void fraction could be determined.
Article
Titanate nanotubes were synthesized from TiO2 in alkaline medium by a conventional hydrothermal method (150 °C, 4.7 bar). To obtain hydrogen titanates, the as-prepared sodium titanates were treated with either HCl or H3PO4 aqueous solutions. A simple synthesis procedure was devised for stable titanate nanotube sols without using any additives. These highly stable ethanolic sols can readily be used to prepare transparent titanate nanotube thin films of high quality. The resulting samples were studied by X-ray diffraction, N2-sorption measurements, Raman spectroscopy, transmission and scanning electron microscopy, X-ray photoelectron spectroscopy and spectroscopic ellipsometry. The comparative results of using two kinds of acids shed light on the superior thermal stability of the H3PO4-treated titanate nanotubes (P-TNTs). X-ray photoelectron spectroscopy revealed that P-TNTs contains P in the near-surface region and the thermal stability was enhanced even at a low (∼0.5 at%) concentration of P. After calcination at 500 °C, the specific surface areas of the HCl- and H3PO4-treated samples were 153 and 244 m2 g−1, respectively. The effects of H3PO4 treatment on the structure, morphology and porosity of titanate nanotubes are discussed.
Article
Multilayer films of organic compounds on solid surfaces have been studied for more than 60 years because they allow fabrication of multicomposite molecular assemblies of tailored architecture. However, both the Langmuir-Blodgett technique and chemisorption from solution can be used only with certain classes of molecules. An alternative approach—fabrication of multilayers by consecutive adsorption of polyanions and polycations—is far more general and has been extended to other materials such as proteins or colloids. Because polymers are typically flexible molecules, the resulting superlattice architectures are somewhat fuzzy structures, but the absence of crystallinity in these films is expected to be beneficial for many potential applications.
Article
Thin polymer films were formed on models of tissue surfaces using polyelectrolyte multilayer techniques, to evaluate the feasibility of using such techniques to build barrier materials onto the surfaces of tissues to improve postsurgical healing, or on the surfaces of tissue-engineered implants. By incubating heterogeneous surfaces with a polycation, followed by a polyanion, layers of polyelectrolyte were deposited onto the surfaces, as confirmed by ellipsometry and water contact angle measurement. Particularly favorable properties were found using the polyelectrolytes polylysine and alginate, which are capable of forming complex gels at physiologic pH; whereas others have demonstrated linear growth in film thickness, with this system, exponential growth was observed under certain conditions, which may be very useful in the coating of heterogeneous surfaces. Surfaces that were treated with multilayer techniques included gelatin, fibroblast extracellular matrix, and fibrillar type I collagen. All surfaces tested were highly heterogeneous and highly adhesive to cells before treatment. The formed thin polymer layers were found to be relatively bioinert, and the thicknesses of the assembles were found to be correlated with bioinertness, such that interactions of cells with the underlying proteinaceous surface could be prevented. The thickness of the polymer layers could be changed by increasing the number of bilayers adsorbed and also by changing the treatment and washing conditions so as to enhance the formation of complex gels.
Article
Generalized ellipsometry and quartz crystal nanogravimetry are combined to determine adsorption isotherms and changes in the optical properties of biaxial TiO2 thin films by monitoring changes in the Mueller matrix. Individual Mueller matrix elements, corresponding to a variety of polarization states, exhibit dramatically different sensitivities to the adsorption of toluene. While some elements are sensitive to structural anisotropy and orientation, others report uniquely on the refractive index. The fast (na) optical axis reflects the greatest change in refractive index due to the adsorption, leading to a decrease from Δn800nm = 0.4 to 0.1. This change is discussed in terms of the Bragg−Pippard (B−P) effective medium approximation, which is shown to accurately describe changes in optical behavior in response to adsorption. The integration of generalized ellipsometry with quartz crystal nanogravimetry establishes a highly sensitive technique for acquiring adsorption isotherms and for chemical optical sensing of structurally anisotropic thin films.Keywords (keywords): glancing angle deposition; reactive ballistic deposition; ellipsometric porosimetry; Mueller matrix; titanium dioxide; anisotropy
Article
Es werden verschiedene physikalische Konstanten heterogener Körper aus den Konstanten ihrer homogenen Bestandteile nach einer einheitlichen Methode berechnet. In dieser ersten Arbeit wird die Berechnung der Dielektrizitätskonstanten und der Leitfähigkeiten für Elektrizität und Wärme der Mischkörper aus isotropen Bestandteilen behandelt. Die Genauigkeit der älteren Formeln wird untersucht und die bis jetzt unbekannten Konstanten dieser Formeln werden berechnet. Sodann wird die Theorie geprüft an Messungen der Leitfähigkeit bei heterogenen Metallegierungen und an den DK. von gepreßten Pulvern und Emulsionen; die verschiedenen Formeln werden bestätigt. Bei dieser Anwendung werden einige Widersprüche zwischen früheren Untersuchungen aufgehoben und es wird versucht, einige ungenau bekannte DK. genauer zu bestimmen.
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
The biosensor based on the total internal reflection imaging ellipsometry (TIRIE) is realized as an automatic analysis method for protein interaction processes in real-time, with high throughput and label-free. An evanescent wave is used as the optical probe to monitor bio-molecular interactions on a chip surface with a high sensitivity due to its phase sensitive property. In this paper, the technique is optimized with a polarization setting, a spectroscopic light source and a low noise CCD detector to improve the performance of the biosensor in sensitivity and detection limit, as evidenced by a quantitative detection of Hepatitis B virus surface antigen (HbsAg) with concentrations of 8, 16, 32, 64, 125 and 250 ng/ml. The sensitivity is increased by one order of magnitude and the detection limit has been extended more than 50 times for HbsAg detection.
Article
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.
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
In this article, spectroscopic ellipsometry studies of plasmon resonances at metal-dielectric interfaces of thin films are reviewed. We show how ellipsometry provides valuable non-invasive amplitude and phase information from which one can determine the effective dielectric functions, and how these relate to the material nanostructure and define exactly the plasmonic characteristics of the system. There are three related plasmons that are observable using spectroscopic ellipsometry; volume plasmon resonances, surface plasmon polaritons and particle plasmon resonances. We demonstrate that the established method of exploiting surface plasmon polaritons for chemical and biological sensing may be enhanced using the ellipsometric phase information and provide a comprehensive theoretical basis for the technique. We show how the particle and volume plasmon resonances in the ellipsometric spectra of nanoparticle films are directly related to size, surface coverage and constituent dielectric functions of the nanoparticles. The regularly observed splitting of the particle plasmon resonance is theoretically described using modified effective medium theories within the framework of ellipsometry. We demonstrate the wealth of information available from real-time in situ spectroscopic ellipsometry measurements of metal film deposition, including the evolution of the plasmon resonances and percolation events. Finally, we discuss how generalized and Mueller matrix ellipsometry hold great potential for characterizing plasmonic metamaterials and sub-wavelength hole arrays. (C) 2011 Elsevier Ltd. All rights reserved.
Article
Ultrathin poly(methyl methacrylate) PMMA films were prepared on gold substrates by spin coating PMMA dissolved in toluene. By varying the concentration of PMMA, spin coating speed and curing condition, we obtained very smooth and ultrathin PMMA films. The PMMA films were transformed into highly reactive film containing carboxylic functionalities using UV/O(3) irradiation. These films were shown to remain stable and reactive for at least one week. We then demonstrated the application of the UV/O(3) treated PMMA films for the detection of microRNAs using a label-free detection method called total internal reflection ellipsometry (TIRE). A limit of detection of 10 pM was established. The technique proposed here is a simple and quick method for generating carboxylic functional films for label-free bioanalytical detection techniques.
Article
Total internal reflection ellipsometry (TIRE) in spectroscopic mode in the wavelength range 400–1200 nm is employed in situ at a solid/liquid interface for investigation of protein adsorption on thin semitransparent gold films. In this configuration, the surface plasmon resonance phenomenon gives a large enhancement of the thin film sensitivity. Adsorption of a monolayer of the protein ferritin is monitored kinetically in situ and results in a change in the ellipsometric parameter Δ of more than 90° compared to 3° in similar ellipsometric measurements on gold substrates. This large sensitivity demonstrates a potential for sensor applications. The ferritin layer optical function is modeled with a Cauchy dispersion model resulting in a layer thickness of 9.2 nm, in good agreement with the dimension of the ferritin molecules. A transition layer between the protein film and the gold layer is necessary to introduce in the model to account for interactions between the protein layer and the gold film. The large sensitivity of TIRE for thin layers opens up a pathway to analyze in detail the structure of thin protein layers provided that a further development of the experimental setup and the model for the protein layer is carried out.
Article
The optical properties of ordered multilayer assemblies produced by alternately dipping a substrate into polycation and polyanion solutions are investigated by using a planar optical waveguide as substrate and measuring the mode indices of the guided waves. The layers show a distinct positive birefringence, from which it is inferred that the polymer chains prefer to be oriented perpendicular to the interface (loops). If the films are equilibrated in water, the anistropy diminishes and the films are somewhat expanded. The kinetics of the assembly process was also studied by arranging for a flux of the polyion solution to pass over the waveguide surface while measuring the mode indices.
Article
Possible applications of polyelectrolyte monolayers and multilayers obtained via layer-by-layer deposition ask for well-defined growth as a function of the number of the adsorption cycles. Essential parameters for the regular adsorption of subsequent polymer layers by electrostatic interactions are the charge of the outermost surface region, the surface of the substrate, and the nature of the polyelectrolyte. Therefore such adsorbed polyelectrolyte layers were characterized by streaming potential measurements in combination with ellipsometric studies. After the adsorption of highly charged polycations such as poly(ethylene imine) on negatively charged surfaces, the surface charge changes: A marked increase of the zeta potential as well as a shift of the isoelectric point is observed for standard polyelectrolytes. These changes depend on the storage of the adsorbed layers under water, or on shear applied in the measuring cell. Only one polyelectrolyte disposing of a large hydrophobic fragments formed stable layers. In this case, no significant changes are observed in dependence on the storage conditions or on shear. By varying the number of polyelectrolytes layers, the dependence of the zeta potential on the pH is found to be identical. Accordingly, the surface charge is nearly independent of the number of layers. The layer thickness in a dried state is determined by variable angle spectroscopic ellipsometry, demonstrating the step-by-step growth of the multilayers.
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 present paper deals with the optimization of metal-clad waveguides for sensor applications to achieve high sensitivity for adlayer and refractive index measurements. By using the Fresnel reflection coefficients both the angular shift and the width of the resonances in the sensorgrams are taken into account. Our optimization shows that it is possible for metal-clad waveguides to achieve a sensitivity improvement of 600% compared to surface-plasmon-resonance sensors. (&COPY; 2004 Elsevier B.V. All rights reserved.
Article
We present an optical labelfree method which combines the advantages of ellipsometry and surface plasmon resonance (SPR). With this surface plasmon enhanced ellipsometry (SPEE) method we measured a giant ellipsometric effect of /nm of an antibody layer. The adequate thickness of the gold layer used for SPEE turned out to be about 25 nm which is significantly less than commonly used for SPR. As typical for SPR we also found a spectral shift of the ellipsometric tan ψ and cos Δ curves to larger wavelengths with increasing refractive index near the gold surface. Restricting to monochromatic light the maximum ellipsometric sensitivity is obtained near to the wavelength where cos Δ changes from negative to positive values. The method yields a sensitivity sufficient to detect changes in the effective thickness of biomolecular layers of less than 10 pm. In comparison to conventional SPR the simultaneously measurement of cos Δ and tan ψ yields more information which is useful for quantitative analysis based on fitting theoretical solutions of Fresnel’s equations to experimental results. In addition, SPEE is less sensitive than traditional SPR to extern stray light and intensity fluctuations of the incident light.
Article
Combinatorial methods, by which large collections or libraries of compounds are synthesized and screened for a particular physical or chemical property, are being applied to inorganic and organic materials including superconductors, phosphors, ferroelectric materials, polymers and catalysts. This technology promises to significantly increase both the efficiency of the materials discovery and optimization process, as well as our information base relating to materials properties.
Article
Recently developed method of Total Internal Reflection Ellipsometry (TIRE) represents a very successful combination of the spectroscopic ellipsometry instrumentation with the Kretchmann type Surface Plasmon Resonance (SPR) geometry of the experiment. The modelling shows much higher sensitivity of the TIRE method to small changes in optical parameters (thickness and refractive index) of thin films, as compared to both traditional external reflection ellipsometry and SPR. Considering another advantage of performing the measurements in media of different optical density (and even opaque media), TIRE becomes very convenient for different sensing applications in both gaseous and liquid media, as well as for thin film characterisation. This work presents examples of applications of the TIRE method for the study of DNA hybridization and the registration of low molecular weight toxins.
Article
The basic theory of off-null ellipsometry is described with emphasis on the determination of the surface concentration of adsorbed proteins on silicon substrates. The coupling between surface concentration and the off-null intensity is found by the use of de Feijters formula. Different optical models for adsorption processes are proposed. For surface concentrations below 5 ng/mm2, they all give a linear relation between the square root of the intensity and the surface concentration of the proteins. The accuracy is of the order of 3% or better. The range of validity of the linearity is discussed using linear expansions of the ellipsometric equations.
Article
Biodegradable polyesters such as poly(lactic-co-glycolic acid) copolymers (PLGA) are preferred materials for drug carrier systems although their surface hydrophobicity greatly limits their use in controlled drug delivery. PLGA thin films on a solid support blended with PEG-containing compound (Pluronic) were used as model systems to study the interfacial interactions with aqueous media. Degree of surface hydrophilization was assessed by wettability, and X-ray photoelectron spectroscopy (XPS) measurements. Protein adsorption behavior was investigated by in situ spectroscopic ellipsometry. The degree of protein adsorption showed a good correlation with the hydrophilicity, and surface composition. Unexpectedly, the layer thickness was found to have a great impact on the interfacial characteristics of the polymer films in the investigated regime (20-200 nm). Thick layers presented higher hydrophilicity and great resistance to protein adsorption. That special behavior was explained as the result of the swelling of the polymer film combined with the partial dissolution of Pluronic from the layer. This finding might promote the rational design of surface modified biocompatible nanoparticles.
Article
Protein adsorption at solid surfaces plays a key role in many natural processes and has therefore promoted a widespread interest in many research areas. Despite considerable progress in this field there are still widely differing and even contradictive opinions on how to explain the frequently observed phenomena such as structural rearrangements, cooperative adsorption, overshooting adsorption kinetics, or protein aggregation. In this review recent achievements and new perspectives on protein adsorption processes are comprehensively discussed. The main focus is put on commonly postulated mechanistic aspects and their translation into mathematical concepts and model descriptions. Relevant experimental and computational strategies to practically approach the field of protein adsorption mechanisms and their impact on current successes are outlined.
Article
A description is given of an ellipsometer, automated by means of computer-steered stepping motors. Conversion of analyzer and polarizer readings into thickness and refractive index is done by a graphic procedure on (Δ,ψ)→(n,d) conversion plots. The method is tested by means of Langmuir-Blodgett monolayers of barium stearate. Adsorption of fibrinogen and albumin onto hydrophilic and hydrophobic chromium surfaces, the interaction of fibrinogen with anti-fibrinogen, and the interaction of albumin with anti-albumin are shown as illustrations of the technique.
Article
This paper presents a comparative study of immobilization strategies for integrated optical grating couplers using tantalum oxide waveguides. As a model system the affinity reaction between protein G and human IgG was investigated. The receptors were coupled to the waveguide by adsorption, covalent attachment and avidin-biotin bridges after modifying the sensor surface by silanization introducing epoxy and amino groups. In addition, the results obtained by a monolayer coverage of the sensors with the receptors were compared with those using a carboxymethyl-dextran matrix allowing the immobilization of an increased amount of protein. The different coupling procedures were assessed by the system response due to the specific binding of human IgG to the sensing layer and are discussed with respect to their effectiveness, stability and the absence of non-specific binding.