Investigating the influence of solvent type and pH on protein adsorption onto silica surface by evanescent-wave cavity ring-down spectroscopy.

Several studies have explored the adsorption of various proteins onto solid-liquid interfaces, revealing the crucial role of buffer solutions in biological processes. However, a comprehensive evaluation of the buffer's influence on protein absorption onto fused silica is still lacking. This study employs evanescent-wave cavity ring-down spectroscopy (EW-CRDS) to assess the influence of buffer solutions and pH on the adsorption kinetics of three globular proteins: hemoglobin (Hb), myoglobin (Mb), and cytochrome c (Cyt-C) onto fused silica. The EW-CRDS tool, with a ring-down time of 1.4 µ s and a minimum detectable absorbance of 1 × 10 - 6 , enabled precise optical measurements at solid-liquid interfaces. The three heme proteins' adsorption behavior was investigated at pH 7 in three different solvents: deionized (DI) water, tris(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl), and phosphate buffered saline (PBS). For each protein, the surface coverage, the adsorption and desorption constants, and the surface equilibrium constant were optically measured by our EW-CRDS tool. Depending on the nature of each solvent, the proteins showed a completely different adsorption trend on the silica surface. The adsorption of Mb on the silica surface was depressed in the presence of both Tris-HCl and PBS buffers compared with unbuffered (DI water) solutions. In contrast, Cyt-C adsorption appears to be relatively unaffected by the choice of buffer, as it involves strong electrostatic interactions with the surface. Notably, Hb exhibits an opposite trend, with enhanced protein adsorption in the presence of Tris-HCl and PBS buffer. The pH investigations demonstrated that the electrostatic interactions between the proteins and the surface had a major influence on protein adsorption on the silica surface, with adsorption being greatest when the pH values were around the protein's isoelectric point. This study demonstrated the ability of the highly sensitive EW-CRDS tool to study the adsorption events of the evanescent-field-confined protein species in real-time at low surface coverages with fast resolution, making it a valuable tool for studying biomolecule kinetics at solid-liquid interfaces.

Adsorption Properties and Electron-transfer Rates of a Redox Probe at Different Interfaces of an Immunoassay Assembled on an Electro-active Photonic Platform.

Physical and chemical properties of a redox protein adsorbed to different interfaces of a multilayer immunoassay assembly were studied using a single-mode, electro-active, integrated optical waveguide (SM-EA-IOW) platform. For each interface of the immunoassay assembly (indium tin oxide, 3-aminopropyl triethoxysilane, recombinant protein G, antibody, and bovine serum albumin) the surface density, the adsorption kinetics, and the electron-transfer rate of bound species of the redox-active cytochrome c (Cyt-C) protein were accurately quantified at very low surface concentrations of redox species (from 0.4 to 4% of a full monolayer) using a highly sensitive optical impedance spectroscopy (OIS) technique based on measurements obtained with the SM-EA-IOW platform. The technique is shown here to provide quantitative insights into an important immunoassay assembly for characterization and understanding of the mechanisms of electron transfer rate, the affinity strength of molecular binding, and the associated bio-selectivity. Such methodology and acquired knowledge are crucial for the development of novel and advanced immuno-biosensors.

Photobleaching reduction in modulated super-resolution microscopy.

Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases in photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.

Electroactive Interface for Enabling Spectroelectrochemical Investigations in Evanescent-Wave Cavity-Ring-Down Spectroscopy.

In this study, we report the development of an electrically active solid-liquid interface for the evanescent-wave cavity-ring-down spectroscopic (EW-CRDS) technique to enable spectroelectrochemical investigations of redox events. Because of a high-quality transparent conductive electrode film of indium tin oxide (ITO) coated on the interface of total internal reflection of the EW-CRDS platform, a cavity ring-down time of about 900 ns was obtained allowing spectroelectrochemical studies at solid-liquid interfaces. As a proof-of-concept on the capabilities of the developed platform, measurements were performed to address the effects of an applied electric potential to the adsorption behavior of the redox protein cytochrome c (Cyt-C) onto different interfaces, namely, bare-ITO, 3-aminopropyl triethoxysilane (APTES), and Cyt-C antibody. For each interface, the adsorption and desorption constants, the surface equilibrium constant, the Gibbs free energy of adsorption, and the surface coverage were optically measured by our electrically active EW-CRDS tool. Optical measurements at a set of constant discrete values of the applied electric potential were acquired for kinetic adsorption analysis. Cyclic voltammetry (CV) scans under synchronous optical readout were performed to study the effects of each molecular interface on the redox process of surface-adsorbed protein species. Overall, the experimental results demonstrate the ability of the electro-active EW-CRDS platform to unambiguously measure electrode-driven redox events of surface-confined molecular species at low submonolayer coverages and at a single diffraction-limited spot. Such capability is expected to open several opportunities for the EW-CRDS technique to investigate a variety of electrochemical phenomena at solid-liquid interfaces.

Detection of influenza virus by electrochemical surface plasmon resonance under potential modulation.

In this study we report the development of a novel viral pathogen immunosensor technology based on the electrochemical modulation of the optical signal from a surface plasmon wave interacting with a redox dye reporter. The device is formed by incorporating a sandwich immunoassay onto the surface of a plasmonic device mounted in a micro-electrochemical flow cell, where it is functionalized with a monoclonal antibody aimed to a specific target pathogen antigen. Once the target antigen is bound to the surface, it promotes the capturing of a secondary polyclonal antibody that has been conjugated with a redox-active methylene blue dye. The methylene blue displays a reversible change in the complex refractive index throughout a reduction-oxidation transition, which generates an optical signal that can be electrochemically modulated and detected at high sensitivity. For proof-of-principle measurements, we have targeted the hemagglutinin protein from the H5N1 avian influenza A virus to demonstrate the capabilities of our device for detection and quantification of a critical influenza antigen. Our experimental results of the EC-SPR-based immunosensor under potential modulation showed a 300 pM limit of detection for the H5N1 antigen.

Influenza virus immunosensor with an electro-active optical waveguide under potential modulation.

Here we report the development of a novel immunosensor-based strategy for label-free detection of viral pathogens by incorporating a sandwich bioassay onto a single-mode, electro-active, integrated optical waveguide (EA-IOW). Our strategy begins with the functionalization of the electro-active waveguide surface with a capture antibody aimed at a specific virus antigen. Once the target antigen is bound to the photonic interface, it promotes the binding of a secondary antibody that has been labeled with a methylene blue (MB) dye. The MB is a redox-active probe whose optical absorption can be electrically modulated and interrogated with high sensitivity by a propagating waveguide mode. In this effort, we have targeted the hemagglutinin (HA) protein from the H5N1 avian influenza A virus to demonstrate the capabilities of the EA-IOW device for detection and quantification of an important antigen. Our initial results for the HA H5N1 influenza virus show a remarkable limit of detection in the pico-molar range.

Electron-Transfer Rate in Potential-Modulated Redox Reactions with Electro-Active Optical Waveguides.

A novel methodology has been developed to determine electron-transfer rate in electrically driven redox reactions. Based on a widely adopted electrical circuit describing faradaic processes in an electrochemical cell, the approach uses a combination of impedance data from optical and electrical measurements that are simultaneously acquired in a spectroelectrochemical experiment. Once the consistency of our methodology was experimentally corroborated, it was put to practice for investigating electron-transfer rate of cytochrome c adsorbates at very low concentrations on an indium tin oxide electrode by using a highly sensitive, single-mode, electro-active, integrated optical waveguide platform. Different surface densities of redox species on the electrode interface and different ionic strengths in the electrolyte solution were studied. Higher surface densities and higher ionic strengths are shown to slow down the electron-transfer process between the redox molecules and the working electrode.

Spectroelectrochemical Properties of Ultra-Thin Indium Tin Oxide Films under Electric Potential Modulation.

In this work, the spectroscopic properties of ultra-thin ITO films are characterized under an applied electric potential modulation. To detect minute spectroscopic features, the ultra-thin ITO film was coated over an extremely sensitive single-mode integrated optical waveguide, which provided a long pathlength with more than adequate sensitivity for optical interrogation of the ultra-thin film. Experimental configurations with broadband light and several laser lines at different modulation schemes of an applied electric potential were utilized to elucidate the nature of intrinsic changes. The imaginary component of the refractive index (absorption coefficient) of the ultra-thin ITO film is unequivocally shown to have a dependence on the applied potential and the profile of this dependence changes substantially even for wavelengths inside a small spectral window (500-600 nm). The characterization technique and the data reported here can be crucial to several applications of the ITO material as a transparent conductive electrode, as for example in spectroelectrochemical investigations of surface-confined redox species.

Surface modification of optical materials with hydrogen plasma for fabrication of Bragg gratings.

We investigate the hydrogen plasma process as a route for creating Bragg gratings (BGs) on optoelectronic materials such as undoped lithium niobate (LiNbO(3)), proton-exchanged LiNbO(3), and soda-lime glass. Photopatterns (periodic modulations, Λ=323-2000 nm) were created on those substrates and the hydrogen plasma process was investigated for its ability to transfer the microstructures and the underlying mechanisms involved in this process. The diffraction efficiency and surface topology of the BG were characterized, as well as the optical properties of corresponding bulk materials undergoing the same plasma treatment. It is shown that the hydrogen plasma treatment changes the complex refractive index and modifies the surface topology with a volume expansion in the near-surface region, and both features are connected to the appearance of structural defects in the materials. The hydrogen plasma offers unique flexibility and advantages that can be explored for the fabrication of integrated photonic components.

Extension of the broadband single-mode integrated optical waveguide technique to the ultraviolet spectral region and its applications.

We report here the fabrication, characterization, and application of a single-mode integrated optical waveguide (IOW) spectrometer capable of acquiring optical absorbance spectra of surface-immobilized molecules in the visible and ultraviolet spectral region down to 315 nm. The UV-extension of the single-mode IOW technique to shorter wavelengths was made possible by our development of a low-loss single-mode dielectric waveguide in the UV region based on an alumina film grown by atomic layer deposition (ALD) over a high quality fused silica substrate, and by our design/fabrication of a broadband waveguide coupler formed by an integrated diffraction grating combined with a highly anamorphic optical beam of large numerical aperture. As an application of the developed technology, we report here the surface adsorption process of bacteriochlorophyll a on different interfaces using its Soret absorption band centred at 370 nm. The effects of different chemical compositions at the solid-liquid interface on the adsorption and spectral properties of bacteriochlorophyll a were determined from the polarized UV-Vis IOW spectra acquired with the developed instrumentation. The spectral extension of the single-mode IOW technique into the ultraviolet region is an important advance as it enables extremely sensitive studies in key characteristics of surface molecular processes (e.g., protein unfolding and solvation of aromatic amino-acid groups under surface binding) whose spectral features are mainly located at wavelengths below the visible spectrum.

Optical impedance spectroscopy with single-mode electro-active-integrated optical waveguides.

An optical impedance spectroscopy (OIS) technique based on a single-mode electro-active-integrated optical waveguide (EA-IOW) was developed to investigate electron-transfer processes of redox adsorbates. A highly sensitive single-mode EA-IOW device was used to optically follow the time-dependent faradaic current originated from a submonolayer of cytochrome c undergoing redox exchanges driven by a harmonic modulation of the electric potential at several dc bias potentials and at several frequencies. To properly retrieve the faradaic current density from the ac-modulated optical signal, we introduce here a mathematical formalism that (i) accounts for intrinsic changes that invariably occur in the optical baseline of the EA-IOW device during potential modulation and (ii) provides accurate results for the electro-chemical parameters. We are able to optically reconstruct the faradaic current density profile against the dc bias potential in the working electrode, identify the formal potential, and determine the energy-width of the electron-transfer process. In addition, by combining the optically reconstructed faradaic signal with simple electrical measurements of impedance across the whole electrochemical cell and the capacitance of the electric double-layer, we are able to determine the time-constant connected to the redox reaction of the adsorbed protein assembly. For cytochrome c directly immobilized onto the indium tin oxide (ITO) surface, we measured a reaction rate constant of 26.5 s(-1). Finally, we calculate the charge-transfer resistance and pseudocapacitance associated with the electron-transfer process and show that the frequency dependence of the redox reaction of the protein submonolayer follows as expected the electrical equivalent of an RC-series admittance diagram. Above all, we show here that OIS with single-mode EA-IOW's provide strong analytical signals that can be readily monitored even for small surface-densities of species involved in the redox process (e.g., fmol/cm(2), 0.1% of a full protein monolayer). This experimental approach, when combined with the analytical formalism described here, brings additional sensitivity, accuracy, and simplicity to electro-chemical analysis and is expected to become a useful tool in investigations of redox processes.

Angle-multiplexed waveguide resonance of high sensitivity and its application to nanosecond dynamics of molecular assemblies.

We report here the experimental demonstration of a high-performance optical waveguide resonance (WR) platform based on a judicious design of a dielectric/metal stack and a fabrication process that delivers an extraordinarily low-loss optical waveguide over a noble-metal thin film. By using an atomic layer deposition process to grow a dielectric film (Al(2)O(3)) of exceptional optical quality and precise thickness over a metal layer (Ag), we have reached a deep and narrow WR that allowed us to experimentally measure a performance of the WR device that is 20 times superior to the conventional surface plasmon resonance sensor. To the best of our knowledge, these results represent the best performance of a WR device reported so far in the literature. In addition, we have created an experimental setup based on diffraction-limited optical components to launch and collect a broad angular spectrum that is able to resolve the sharp angular waveguide resonance at a fast pace. Such configuration has enabled us to reach nanosecond time scale resolution, and we provide here experimental evidence of the fast coupling of the optical signal from a submonolayer of a ruthenium complex adsorbed to the interrogation surface. The high sensitivity and nanosecond detection capability of the WR optical platform demonstrated here are expected to find useful applications for researchers interested in studies of surface-mediated molecular interactions and interfacial phenomena.

Solid immersion lens at the aplanatic condition for enhancing the spectral bandwidth of a waveguide grating coupler.

We report a technique to substantially boost the spectral bandwidth of a conventional waveguide grating coupler by using a solid immersion cylindrical lens at the aplanatic condition to create a highly anamorphic beam and reach a much larger numerical aperture, thus enhancing the spectral bandwidth of a free-space propagating optical beam coupled into a single-mode planar integrated optical waveguide (IOW). Our experimental results show that the broadband IOW spectrometer thus created almost doubles (94% enhancement) the coupled spectral bandwidth of a conventional configuration. To exemplify the benefits made possible by the developed approach, we applied the technique to the broadband spectroscopic characterization of a protein submonolayer; our experimental data confirm the enhanced spectral bandwidth (around 380-nm) and illustrate the potentials of the developed technology. Besides the enhanced bandwidth, the broadband coupler of the single-mode IOW spectrometer described here is more robust and user-friendly than those previously reported in the literature and is expected to have an important impact on spectroscopic studies of surface-adsorbed molecular layers and surface phenomena.

Low-Loss Optical Waveguides for the Near Ultra-Violet and Visible Spectral Regions with Al(2)O(3) Thin Films from Atomic Layer Deposition.

In this work, we report low-loss single-mode integrated optical waveguides in the near ultra-violet and visible spectral regions with aluminum oxide (Al(2)O(3)) films using an atomic layer deposition (ALD) process. Alumina films were deposited on glass and fused silica substrates by the ALD process at substrate/chamber temperatures of 200 °C and 300 °C. Transmission spectra and waveguide measurements were performed in our alumina films with thicknesses in the range of 210 - 380 nm for the optical characterization. Those measurements allowed us to determine the optical constants (n(w) and k(w)), propagation loss, and thickness of the alumina films. The experimental results from the applied techniques show good agreement and demonstrate a low-loss optical waveguide. Our alumina thin-film waveguides is well transparent in the whole visible spectral region and also in an important region of the UV; the measured propagation loss is below 4 dB/cm down to a wavelength as short as 250 nm. The low propagation loss of these alumina guiding films, in particular in the near ultra-violet region which lacks materials with high optical performance, is extremely useful for several integrated optic applications.

An electroactive fiber optic chip for spectroelectrochemical characterization of ultra-thin redox-active films.

The first, fully integrated, planar fiber optic platform with spectroelectrochemical capabilities, termed the electroactive fiber optic chip (EA-FOC) is presented here. Spectroelectrochemical techniques provide complementary optical and electrochemical data which are important for applications ranging from thin film characterization to advanced sensor design. To create the EA-FOC a side-polished fiber optic is coated with a thin film of indium-tin oxide (ITO) as the working electrode and used to probe electrochemically-driven changes in absorbance for surface-confined redox species. A sensitivity enhancement of approximately 40 times higher than a transmission measurement is demonstrated for this first-generation EA-FOC, using the methylene blue (MB) redox couple, cycling between the visibly colored, oxidized form of MB, and its leuco (transparent) reduced form. Additionally, the EA-FOC is used to probe the redox spectroelectrochemistry of an electrodeposited thin film, about 0.3% of a monolayer, of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT). Unlike other waveguide formats, the EA-FOC offers an ease of use due to its ability to simply couple to light sources and detectors through standard fiber connectors to create a sensitive planar waveguide spectroelectrochemical platform.

Investigations on the Q and CT Bands of Cytochrome c Submonolayer Adsorbed on an Alumina Surface Using Broadband Spectroscopy with Single-Mode Integrated Optical Waveguides.

In this work, we report experimental results on the molar absorptivity of cytochrome c adsorbed at different submonolayer levels onto an aluminum oxide waveguide surface; our data show a clear dependence of the protein optical properties on its surface density. The measurements were performed using the broadband, single-mode, integrated optical waveguide spectroscopic technique, which is an extremely sensitive tool able to reach submonolayer levels of detection required for this type of studies. This investigation focuses on the molar absorptivity at the Q-band (centered at 525 nm) and, for the first time to our knowledge, the weak charge transfer (CT) band (centered at 695 nm) of surface-adsorbed cyt c. Polarized light in the spectral region from 450 to 775 nm was all-coupled into an alumina thin film, which functioned as a single-mode planar optical waveguide. The alumina thin-film waveguide used for this work had a thickness of 180 nm and was deposited on a glass substrate by the atomic layer deposition process. The protein submonolayer was formed on the alumina waveguide surface through electrostatic adsorption from an aqueous buffer solution at neutral pH. The optical properties of the surface-adsorbed cyt c were investigated for bulk protein concentrations ranging from 5 nM to 8200 nM in the aqueous buffer solution. For a protein surface density of 2.3 pmol/cm(2), the molar absorptivity measured at the charge transfer band was 335 M(-1) cm(-1), and for a surface density of 15 pmol/cm(2) was 720 M(-1) cm(-1), which is much closer to the value of cyt c dissolved in an aqueous neutral buffer (830 M(-1) cm(-1)). The modification of the protein molar absorptivity and its dependence on the surface density can most likely be attributed to conformational changes of the surface-adsorbed species.

Planar fiber-optic chips for broadband spectroscopic interrogation of thin films.

A planar fiber-optic chip (FOC) has been developed using side-polished optical fibers and characterized for broadband absorbance and fluorescence detection of molecular films. FOC technology combines the sensitivity of an attenuated total reflection (ATR) element with the ease of use of fiber-optic-based spectrometers and light sources to create an improved platform for spectroscopic analysis of molecular adsorbates. A multi-mode optical fiber (core diameter = 50 mum, numerical aperture = 0.22, stepped refractive index profile) mounted in a glass V-groove block was side-polished to create a planar platform that allows access to the evanescent field escaping from the fiber core. For this generation of FOC technology, the exposed evanescent field has an interaction length of approximately 17.2 mm. The FOC platform was independently characterized through measurements of thin-film and bulk absorbing samples. The device performance was compared to the existing ATR technology and methods for increasing sensitivity of the FOC were investigated and validated. Additionally, we have demonstrated the ability of the FOC to both evanescently excite and collect fluorescence through guided modes of the optical fiber for a surface-confined luminescent semiconductor nanoparticle film (4 nm diameter, ligand capped, CdSe core). The FOC described here with a supported planar interface can facilitate the use of conventional planar deposition technologies and provide a robust planar platform that is amenable for incorporation into various sensor technologies.

Highly sensitive spectroscopic detection of heme-protein submonolayer films by channel integrated optical waveguide.

A highly sensitive technique based on optical absorption using a single-mode, channel integrated optical waveguide is described for broad spectral band detection and analysis of heme-containing protein films at a glass/water interface. Fabrication steps and device characteristics of optical waveguides suitable for operation in the wavelength range of 400 - 650 nm are described. Experimental results reported here show a limit of detection smaller than 100 pg/cm(2) for a submonolayer of ferricytochrome c by probing the Soret transition band with a 406-nm semiconductor diode laser propagating in a single-mode, ion-exchanged channel waveguide. By taking advantage of the exceptionally low limit of detection, we examined the adsorption isotherm of cytochrome c on a glass surface with unprecedented detail. Unlike other surface-specific techniques (e.g. SPR, integrated optic Mach-Zehnder interferometer) that probe local refractive-index changes and therefore are very susceptible to temperature fluctuations, the integrated optical waveguide absorption technique probes molecular-specific transition bands and is expected to be less vulnerable to environmental perturbations.

Combination of polarized TIRF and ATR spectroscopies for determination of the second and fourth order parameters of molecular orientation in thin films and construction of an orientation distribution based on the maximum entropy method.

This article describes two mathematical formalisms for the determination of the second and fourth order parameters of molecular films using optical spectroscopy. Method A uses polarized total internal reflection fluorescence (TIRF) to calculate the second and fourth order parameters, {P2(cos theta)} and {P4(cos theta)}, using an independently determined value for the angle between the absorption and emission dipoles, gamma. Method B uses {P2(cos theta)} obtained from attenuated total reflectance (ATR) data, along with polarized TIRF measurements to calculate {P4(cos theta)} and {cos2 gamma}. The choice of a specific method should rely on experimental considerations. We also present a method to separate the contributions of substrate surface roughness and dipole orientation with respect to the molecular axis from the spectroscopically determined second and fourth order parameters. Finally, a maximum entropy approach for construction of an orientation distribution from order parameters is compared with the commonly used delta and Gaussian distributions.

Order parameters and orientation distributions of solution adsorbed and microcontact printed cytochrome c protein films on glass and ITO.

The structure of solution adsorbed and microcontact printed (muCP) cytochrome c (cyt c) films on glass and indium tin oxide (ITO) was investigated using attenuated total reflectance (ATR) and total internal reflectance fluorescence (TIRF) spectroscopies to determine the orientation of the heme groups in the films. The second and fourth order parameters of the heme as well as information on the angle between the absorption and emission dipoles of the heme, gamma, were experimentally determined. The order parameters of the heme are related to the order parameters of the protein molecule using the known angle between the heme plane and the electrostatic dipole moment of the cyt c protein. The effect of the surface roughness of the substrates (glass and ITO) was also taken into account quantitatively using AFM data. Physically possible order parameters were obtained for the heme group in both solution adsorbed and muCP films, but not for the electrostatic dipole moment of the protein. In addition, the experimental values of {cos2 gamma} for immobilized zinc-substituted cyt c are greater than the values of {cos2 gamma} determined in viscous solutions, which could be an indication that the environment of the heme groups changes upon adsorption. The electron transfer behavior of solution adsorbed and muCP films on ITO, determined using electrochemical methods, is compared to their orientation distribution and surface coverage as determined by spectroscopic methods.