Measurement of film thickness up to several hundreds of nanometers using optical waveguide lightmode spectroscopy

Up to now, most studies based on optical waveguide lightmode spectroscopy (OWLS) were dedicated to thin adlayers, assumed to be isotropic and homogeneous, for which data analysis was based on an approximation of the mode equations valid when the thickness is small with respect to the wavelength of the laser light. The aim of the present paper is to extend the use of OWLS to thicker deposited layers (up to approximately 400 nm). Both the simplified and extended models are compared in terms of optical parameters, i.e. the refractive index nA, the thickness dA, and the optical mass QA, for experimental data obtained with polyelectrolyte multilayer films. The deviation of these parameters can be quite large when derived using the simplified model instead of the extended model. This observation evidences that OWLS is well suited for the study of "thick" films if the appropriate model is applied to the data analysis.     

Multidepth screening of living cells using optical waveguides

The use of planar optical waveguides as substrata for label-free, non-invasive monitoring of cells growing on them is demonstrated. Different submicrometre depths (measured from and perpendicular to the substratum surface) can be selected for monitoring. The so-called symmetry waveguide configuration with a low refractive index waveguide support (nanoporous silica with refractive index approximately 1.2) and a polystyrene waveguiding film with a heat-embossed grating coupler is exploited to obtain practically useful differences between the penetration depths of different waveguide modes. Robust data processing techniques are developed to obtain quantitative information about the cell refractive index profile perpendicular to the substratum from the measured effective refractive indices of the modes. In particular, a method is introduced with which cell refractive index variations above and below a predefined and tunable depth can be separated using two modes. The technique can be extended to more modes to gain even more comprehensive information from predefined submicrometre slices of the cell layer. The introduced methods are also suitable for monitoring the kinetics of changes in cell refractive index profiles.     

Model experiments with integrated optical input grating couplers as direct immunosensors

Using an input grating coupler instrument and monomode optical waveguides, we studied the adsorption of various proteins (e.g. human immunoglobulins G and A (h-IgG and h-IgA), and avidin) on the waveguide surface. In model experiments for immunoassays, we also monitored as a function of time the formation of time formation of the immuno-complex Ag-Ab between antigen (Ag) molecules absorbed on the waveguide surface and antibody (Ab) molecules, for example, of adsorbed h-IgG vs anti-h-IgG, anti-goat-IgG (cross-reaction) and anti-h-IgA (negative test). We also studied the affinity reaction between adsorbed protein A and h-IgG. From the measured changes in the effective refractive indices N(TE0) and N(TM0) of the TE0 and TM0 modes, we determined the thickness dF, the refractive index nF, and the surface coverage gamma of the absorbed protein adlayers and of the immuno-complexes.     

Preparation and optical parameter characterization of two aldehyde derivative thin films for photonic applications by drop casting method

In this study, thin films of polymer poly(methyl methacrylate) were prepared using a drop casting method. Two newly synthesized aldehyde derivatives, 2‐bromomalonaldehyde and 5,6‐dihydroimidazo[2,1‐b]thiazole‐2‐carbaldehyde, were used at different concentrations to dope the films. The prepared films were transparent and therefore studied for application in photonics. Optical characterization of the samples was carried out using different spectroscopy techniques. Absorption spectra for both samples were obtained using a UV–vis light spectrophotometer. Other significant optical parameters, such as refractive index, extinction coefficient, and band gap energies, were calculated from the absorption spectra. The effect of doping concentration on these parameters was studied. Emission spectra were obtained using a fluorescence spectrophotometer and the effect of doping was observed. Fourier transform infrared spectra of the doped films were obtained and compared with the pure compound to note changes in peak values and peak intensity. This present work studied the effect of doping on optical properties and examined the application of the samples for photonics.     

Analysis of the response of planar polarization interferometer to molecular layer formation: fibrinogen adsorption on silicon nitride surface.

The most sensitive optical method of interferometry was exploited for determination of changes in the refractive index following the adsorption of biological molecules onto the solid surface. Instead of having two waveguiding arms (the main and the reference) in traditional Mach-Zhender interferometer, two ortogonal TM and TE modes propagating through the SiO(2)-Si(3)N(4)-SiO(2) waveguide structure were employed in planar polarization interferometer (PPI). Multiperiodic PPI response was, therefore, formed due to the phase shift between TM and TE modes. A matrix simulation procedure was developed in order to investigate the influence of both the refractive index and molecular layer thickness on the PPI response. Nonspecifical binding of fibrinogen to silicon nitride surface was studied as a model object for PPI testing. The results obtained are in good agreement with the known information about fibrinogen adsorption on the different surfaces. An attempt to introduce the concept of 'surface molecular concentration and molecular polariziability' instead of 'molecular layer thickness and refractivity' was undertaken.     

Quantification of biofilm accumulation by an optical approach

Methods for non-invasive, in situ, measurements of biofilm optical density and biofilm optical thickness were evaluated based on Pseudomonas aeruginosa experiments. Biofilm optical density, measured as intensity reduction of a light beam transmitted through the biofilm, correlates with biofilm mass, measured as total carbon and as cell mass. The method is more sensitive and less labor intensive than other commonly used methods for determining extent of biofilm mass accumulation. Biofilm optical thickness, measured by light microscopy, is translated into physical thickness based on biofilm refraction measurements. Biofilm refractive index was found to be close to the refractive index of water. The P. aeruginosa biofilms studied reached a pseudo steady state in less than a week, with stable liquid phase substrate, cell and TOC concentrations and average biofilm thickness. True steady state was, however, not reached as both biofilm density and roughness were still increasing after 3 weeks.     

Application of the optical waveguide lightmode spectroscopy to monitor lipid bilayer phase transition

An instrument for optical waveguide lightmode spectroscopy (OWLS) was designed and developed for measurements at different and controlled temperatures in a range of 15 degrees C around room temperature. The instrument allows to scan the waveguide modes at different wavelengths on the same optical chip using different lasers. This instrument was used to monitor DMPC lipid bilayer main phase transition around the critical temperature. The main problem in these experiments is that the OWLS measurements do not give enough information about an optically anisotropic system like a lipid bilayer. Experimental OWLS data at two different wavelengths can however approximately solve the problem. The temperature dependence of the thickness and the refractive indices (ordinary and extraordinary) for the lipid bilayer around the phase transition is presented. (A theoretical derivation of the extraordinary refractive index is given in.)     

The effect of UV irradiation on uracil thin layer measured by optical waveguide lightmode spectroscopy

The polycrystalline uracil thin-layer dosimeter is a well-established method to monitor the biological effects of the environmental ultraviolet (UV) radiation. It is based on the optical density (OD) decrease of the uracil layer in the UV absorption band due to photodimerization of the crystal caused by UV irradiation. In the present study, we report measurements made with optical waveguide lightmode spectroscopy (OWLS) to characterize the changes in the optogeometrical parameters of the uracil layer caused by an artificial UV source. It is shown that UV irradiation causes a decrease in the refractive index and an increase of the optical anisotropy. The determined kinetic parameters of the UV dose-sensor response curves correlate well with results of OD measurements, but the sensitivity of OWLS is about ten times higher. The results show that OWLS is capable of analyzing the UV response of the uracil layer and opens the way for dosimetrical applications.     

Binding of agonists, antagonists and inverse agonists to the human delta-opioid receptor produces distinctly different conformational states distinguishable by plasmon-waveguide resonance spectroscopy.

Structural changes induced by the binding of agonists, antagonists and inverse agonists to the cloned delta-opioid receptor from human brain immobilized in a solid-supported lipid bilayer were monitored using plasmon-waveguide resonance (PWR) spectroscopy. Agonist (e.g. deltorphin II) binding causes an increase in membrane thickness because of receptor elongation, a mass density increase due to an influx of lipid molecules into the bilayer, and an increase in refractive index anisotropy due to transmembrane helix and fatty acyl chain ordering. In contrast, antagonist (e.g. TIPPpsi) binding produces no measurable change in either membrane thickness or mass density, and a significantly larger increase in refractive index anisotropy, the latter thought to be due to a greater extent of helix and acyl chain ordering within the membrane interior. These results are closely similar to those reported earlier for another agonist (DPDPE) and antagonist (naltrindol) [Salamon et al. (2000) Biophys. J.79, 2463-2474]. In addition, we now find that an inverse agonist (TMT-Tic) produces membrane thickness, mass density and refractive index anisotropy increases which are similar to, but considerably smaller than, those generated by agonists. Thus, a third conformational state is produced by this ligand, different from those formed by agonists and antagonists. These results shed new light on the mechanisms of ligand-induced G-protein-coupled receptor functioning. The potential utilization of this new biophysical method to examine structural changes both parallel and perpendicular to the membrane normal for GPCRs is emphasized.     

The study of genomic DNA adsorption and subsequent interactions using total internal reflection ellipsometry

The adsorption of genomic DNA and subsequent interactions between adsorbed and solvated DNA was studied using a novel sensitive optical method of total internal reflection ellipsometry (TIRE), which combines spectroscopic ellipsometry with surface plasmon resonance (SPR). Single strands of DNA of two species of fish (herring and salmon) were electrostatically adsorbed on top of polyethylenimine films deposited upon gold coated glass slides. The ellipsometric spectra were recorded and data fitting utilized to extract optical parameters (thickness and refractive index) of adsorbed DNA layers. The further adsorption of single stranded DNA from an identical source, i.e. herring ss-DNA on herring ss-DNA or salmon ss-DNA on salmon ss-DNA, on the surface was observed to give rise to substantial film thickness increases at the surface of about 20-21 nm. Conversely adsorption of DNA from alternate species, i.e. salmon ss-DNA on herring ss-DNA or herring ss-DNA on salmon ss-DNA, yielded much smaller changes in thickness of 3-5 nm. AFM studies of the surface roughness of adsorbed layers were in line with the TIRE data.     

Silicon photonic crystal nanocavity-coupled waveguides for error-corrected optical biosensing

A photonic crystal (PhC) waveguide based optical biosensor capable of label-free and error-corrected sensing was investigated in this study. The detection principle of the biosensor involved shifts in the resonant mode wavelength of nanocavities coupled to the silicon PhC waveguide due to changes in ambient refractive index. The optical characteristics of the nanocavity structure were predicted by FDTD theoretical methods. The device was fabricated using standard nanolithography and reactive-ion-etching techniques. Experimental results showed that the structure had a refractive index sensitivity of 10(-2) RIU. The biosensing capability of the nanocavity sensor was tested by detecting human IgG molecules. The device sensitivity was found to be 2.3±0.24×10(5) nm/M with an achievable lowest detection limit of 1.5 fg for human IgG molecules. Additionally, experimental results demonstrated that the PhC devices were specific in IgG detection and provided concentration-dependent responses consistent with Langmuir behavior. The PhC devices manifest outstanding potential as microscale label-free error-correcting sensors, and may have future utility as ultrasensitive multiplex devices.     

Facile fabrication of gelatin‐based biopolymeric optical waveguides

The rapid development in optical detection techniques for sensing applications has led to an increased need for biocompatible, biodegradable, and disposable optical components. We present a controllable fabrication technique for an entirely biopolymeric planar optical waveguide via simple spin‐coating. The refractive index difference, thermal responsive properties, and inherent biocompatibility of gelatin and agarose were exploited in the fabrication of thin, stacked films that efficiently guide light in a core layer with higher index of refraction. These planar waveguides were fabricated using a simple spin‐coating technique, which resulted in controllable layer thicknesses and smooth layer interfaces. This technique, therefore, offers a path for routine engineering of biopolymer structures with contrasting refractive indices. The thermal stability of the gelatin core layer was improved using two crosslinkers; glutaraldehyde or microbial Transglutaminase. Light guiding in the core layer of the waveguide was demonstrated using a simple He–Ne laser setup. Guiding efficiency was further illustrated by directly embedding fluorescent markers within the core layer and detecting their spectral signature. Combined with the biopolymers' inherent biocompatibility and biodegradability, our simple strategy to fabricate disposable optical components holds the potential for the development of applications in biological sensing and implantable biomedical devices. Biotechnol. Bioeng. 2009;103: 725–732. © 2009 Wiley Periodicals, Inc.     

Plasmon-waveguide resonance and impedance spectroscopy studies of the interaction between penetratin and supported lipid bilayer membranes

The interaction between the cell-penetrating peptide, penetratin, and solid-supported lipid bilayer membranes consisting of either egg phosphatidylcholine (PC) or a 75/25 mol% mixture of egg PC and palmitoyloleylphosphatidylglycerol has been studied by simultaneously measuring plasmon-waveguide resonance (PWR) spectra and impedance spectra of lipid-peptide mixtures. When penetratin was incorporated into an egg PC + palmitoyloleylphosphatidylglycerol bilayer, PWR measurements showed a hyperbolic increase in the average refractive index and the refractive index anisotropy, with no change in membrane thickness, over a concentration range between 0 and 2 micro M peptide. In the case of an egg PC bilayer, a biphasic dependence was observed, with a decrease in average refractive index and anisotropy and no thickness change occurring between 0 and 5 micro M peptide, and an increase in membrane thickness occurring between 5 and 15 micro M peptide with no further change in the refractive index parameters. For both membranes, the impedance spectroscopy measurements demonstrated that the electrical resistance was not altered by peptide incorporation, whereas a decrease in membrane capacitance occurred with the same concentration dependence as observed in the PWR experiments, although for the PC membrane no further changes in electrical properties were observed in the higher concentration range. A structural interpretation of these results is described, in which the peptide binds electrostatically within the headgroup region of the bilayer and influences the headgroup conformation, amount of bound water, and the lipid-packing density, without perturbing the hydrocarbon core of the bilayer.     

Biosensing under an applied voltage using optical waveguide lightmode spectroscopy

An applied dc voltage offers a means of controlling immobilization during biosensor fabrication and detection during biosensing application. We present a method to directly and continuously measure the adsorption of biomacromolecules or other polyelectrolytes, under an applied potential difference, based on optical waveguide lightmode spectroscopy (OWLS). An indium tin oxide (ITO) film of thickness ca. 10 nm coated onto a silicon titanium oxide (STO) waveguiding film serves as the working (sensing) electrode. We observe the effective refractive index of the 0th transverse electric guided mode to increase significantly in the presence of an applied potential due to charging of the interfacial double layer and, possibly, modest electrochemical oxidation. Adsorption from solution onto the ITO electrode is detected by a further increase in the effective refractive index. We achieve accurate detection by employing an optical model in which the STO and ITO layers are combined into a single waveguiding film. No improvement is found using models treating the ITO as a separate layer, either dielectric or conducting. Using this method, we find the adsorption of human serum albumin and horse heart cytochrome c to be considerably enhanced in the presence of an applied potential exceeding 1 V. We attribute this behavior to adsorption at positions on the protein molecules of complementary charge.     

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field induced conformational changes of bacteriorhodopsin were studied in six types of dried film (randomly and electrically oriented membranes of purple as well as cation-depleted blue bacteriorhodopsin) by measuring the frequency dependence of the optical absorbance change and the dielectric dispersion and absorption. For the purple bacteriorhodopsin the optical absorbance change induced by alternating rectangular electric fields of ±300 kV/cm altered the sign twice in the frequency range from 0.001 Hz to 100 kHz (around 0.03 Hz and 100 kHz), indicating that the electric field induced conformational change in these samples consists of, at least, three steps. Similarly, it was found for the blue bacteriorhodopsin that at least two steps are involved. In accord with optical measurements, the dielectric behaviour due to alternating sinusoidal electric fields of±6kV/cm in the frequency range from 10 Hz to 10 MHz showed two broad dispersion/absorption regions, one below 1 kHz and the other around 10–100 kHz. This suggests that the conformational change of bacteriorhodopsin is also reflected by its dielectrical properties and that it is partially induced at 6 kV/cm. Including previous results obtained by analysis of the action of DC fields on purple membrane films, a model for a field-induced cyclic reaction for purple as well as blue bacteriorhodopsin is proposed. In addition it was found that there are electrical interactions among purple membrane fragments in dried films.     

Structurally colored thin films of Ca2+-cross-linked alginate

Alginate, or alginic acid, is an unbranched binary copolymer of (1-->4)-linked beta-D-mannuronic acid and alpha-L-guluronic acid. Alginate readily forms binding interactions with a variety of divalent metal ions, such as calcium. This binding has been used to cross-link bulk alginates for a wide variety of applications, particularly in areas of tissue engineering, medical devices, and wound-healing dressings. A new method is identified here for producing Ca2+-cross-linked thin films of sodium alginate, using an aerosolized spray of CaCl2 solution. These thin films exhibit structural color that varies with film thickness. It is demonstrated that this structural color is highly reproducible and can also be tuned to produce a wide range of colored films. The noted ability of alginates to bind metal ions is used in combination with the structural coloration afforded by the thin film structure as a basis for color-based optical sensing of metal ions in aqueous solutions. Changes in film thickness, refractive index, and reflectivity in response to metal ions have been measured and reported. For certain ions such as Cr(III) and Cr(VI), changes in film thickness are the predominate factors in shifting the reflected film color. In the case of other ions such as Pb(II), a change in film refractive index plays a significant role in the reflectance properties of films.     

Towards a biosensor based on anti resonant reflecting optical waveguide fabricated from porous silicon

Recently, we demonstrated that Anti Resonant Reflecting Optical Waveguide (ARROW) based on porous silicon (PS) material can be used as a transducer for the development of a new optical biosensor. Compared to a conventional biosensor waveguide based on evanescent waves, the ARROW structure is designed to allow a better overlap between the propagated optical field and the molecules infiltrated in the porous core layer and so to provide better molecular interactions sensitivity. The aim of this work is to investigate the operating mode of an optical biosensor using the ARROW structure. We reported here an extensive study where the antiresonance conditions were adjusted just before the grafting of the studied molecules for a given refractive index range. The interesting feature of the studied ARROW structure is that it is elaborated from the same material which is the porous silicon obtained via a single electrochemical anodization process. After oxidation and preparation of the inner surface of porous silicon by a chemical functionalization process, bovine serum albumin (BSA) molecules, were attached essentially in the upper layer. Simulation study indicates that the proposed sensor works at the refractive index values ranging from 1.3560 to 1.3655. The experimental optical detection of the biomolecules was obtained through the modification of the propagated optical field and losses. The results indicated that the optical attenuation decreases after biomolecules attachment, corresponding to a refractive index change Δn(c) of the core. This reduction was of about 2 dB/cm and 3 dB/cm for Transverse Electric (TE) and Transverse Magnetic (TM) polarizations respectively. Moreover, at the detection step, the optical field was almost located inside the core layer. This result was in good agreement with the simulated near field profiles.     

Real-time study of the effect of different stress factors on lactic acid bacteria by electrochemical optical waveguide lightmode spectroscopy

Lactic acid bacteria play an important role in the fermentation of different food products. During the fermentation processes, lactobacilli are confronted with many inhibitor factors. These factors by themselves or in combination can influence the growth of lactic acid bacteria and their acidification capacity. The subject of our study was to monitor with a newly developed biosensing technique how the different chemical stress factors influence the survival of lactic acid bacteria. Electrochemical optical waveguide lightmode spectroscopy combines evanescent-field optical sensing with electrochemical control of surface adsorption processes. For optical sensing, a layer of indium tin oxide served as a high refractive index waveguide and as a conductive electrode, as well. Lactobacillus plantarum 2142 suspended in Jerusalem artichoke syrup was used in the experiments. Electrochemical optical waveguide lightmode spectroscopy measurements were undertaken by using OW 2,400c indium tin oxide coated waveguide sensors (MicroVacuum, Budapest, Hungary) and were performed in a flow-injection analyzer system. The bacterial cells were adsorbed in native form without any chemical binding on the surface of the sensor by ensuring polarizing potential (1V) and were exposed to different concentration of acetic acid/Jerusalem artichoke syrup, lactic acid/Jerusalem artichoke syrup and hydrogen peroxide/Jerusalem artichoke syrup solution for 1h, respectively, and the effect on bacteria cells was monitored. Results were compared to the traditional micro-assay method, and it can be assumed that after further investigations this new technique could be used in real-time application.     

A new quantitative optical biosensor for protein characterisation

A new optical biosensor is described based on a dual waveguide interferometric technique. By addressing the waveguide structure with alternate polarisations the optogeometrical properties (density and thickness) of adsorbed protein layers at the sensor (solid)-liquid interface have been determined. Differences in the waveguide mode dispersion between the transverse electric (TE) and transverse magnetic (TM) modes allow unique solutions for adlayer thickness and refractive index to be determined at all stages during the formation process. The technique has been verified using standard protein systems and by comparing the data with published work using X-ray crystallography and neutron reflection techniques. The data obtained was found to be in excellent agreement with previously reported X-ray experiments given that typical film thicknesses for streptavidin layers were in the range 5.5-6.5 nm compared with the short axis crystal structure of between 4.8 and 5.6 nm. The precision of the measurements taken was of the order of 40 pm with respect to adsorbed adlayer thicknesses. This biosensor approach provides measurements of both thickness and density of adlayers to a high precision, simultaneously and in real time enabling detail of the structure and function of proteins to be elucidated. From such data it is possible to obtain information on the orientation, distortion and efficiency of immobilisation procedures as well as the interaction event of interest. The technique is expected to find utility with those interested in protein structure and function. This is an area of growing importance within the life sciences as the demand for quantitative analytical techniques increases with the growth in "proteomics".     

Thickness and density of protein films by optical mixing spectroscopy.

The adsorption of protein films on polystyrene latex spheres was studied by optical mixing spectroscopy. With this technique, we show that both the hydrodynamic thickness of protein films and their optical density can be measured. Thus, we found that films of the glycoproteins isolated from the human erythrocyte membrane were four times as thick as films of either human serum albumin or bovine serum albumin for about the same surface coverage. This result suggests an end-on orientation for the adsorbed glycoprotein molecules, which is consistent with the model proposed by others for the orientation of these molecules at the surface of the red blood cell itself.