Total internal reflection ellipsometry as a label-free assessment method for optimization of the reactive surface of bioassay devices based on a functionalized cycloolefin polymer

We report a label-free optical detection technique, called total internal reflection ellipsometry (TIRE), which can be applied to study the interactions between biomolecules and a functionalized polymer surface. Zeonor (ZR), a cycloolefin polymer with low autofluorescence, high optical transmittance and excellent chemical resistance, is a highly suitable material for optical biosensor platforms owing to the ease of fabrication. It can also be modified with a range of reactive chemical groups for surface functionalization. We demonstrate the applications of TIRE in monitoring DNA hybridization assays and human chorionic gonadotrophin sandwich immunoassays on the ZR surface functionalized with carboxyl groups. The Ψ and Δ spectra obtained after the binding of each layer of analyte have been fitted to a four-layer ellipsometric model to quantitatively determine the amount of analytes bound specifically to the functionalized ZR surface. Our proposed TIRE technique with its very low analyte consumption and its microfluidic array format could be a useful tool for evaluating several crucial parameters in immunoassays, DNA interactions, adsorption of biomolecules to solid surfaces, or assessment of the reactivity of a functionalized polymer surface towards a specific analyte.     

Investigation of rapid thermally annealed GaP(001) surfaces in vacuum

Morphology of rapid thermally annealed GaP(001) surfaces has been investigated using spectroscopic ellipsometry (SE), optical microscopy, ex situ atomic force microscopy, electron probe microanalysis (EPMA) and X‐ray photoelectron spectroscopy (XPS). The samples were annealed in vacuum for t = 2 s at temperatures T = 20–900 °C. The SE, optical microscopy and XPS spectra suggest that thermal annealing causes little influence on the GaP surface at T ≤ 600 °C; however, micro‐ and macroscopic roughening occur at T > 600 °C and T ≥ 750 °C, respectively, with a generation of Ga droplets at T ≥ 750 °C. The presence of the Ga droplets is confirmed by the EPMA measurements. The droplet density can be expressed as N_Ga ∝ exp (E_a/k_BT) with an activation energy of E_a ～ 2.3 eV. The XPS data indicate the change in the surface oxide composition from the native oxide to the Ga oxide (Ga₂O₃ and Ga₂O) after annealing at T ≥ 750 °C. Possible annealing‐induced degradation steps are proposed to provide as complete a picture as possible. Copyright © 2010 John Wiley & Sons, Ltd.     

Infrared spectroscopic ellipsometry (IRSE) and X‐ray photoelectron spectroscopy (XPS) monitoring the preparation of maleimide‐functionalized surfaces: from Au towards Si (111)

The IR ellipsometric technique was used to identify the surface species and to control the preparation of maleimide‐terminated surfaces. Because of higher s/n ratios for metallic substrates, the protocol was initially developed on Au surfaces, was later successfully transferred to technologically more relevant Si (111) substrates. The functionalized surfaces were achieved by electrochemical deposition of diazonium linker films and following chemical adsorption steps. Complementary XPS was also employed to detect the surface species in the process of preparation. The immobilization of different functional molecules was proven by interpreting the specific vibrational bands in IR spectra and additionally confirmed by XPS experiments. The surface homogeneity was investigated by FT‐IR synchrotron mapping ellipsometry. This work shows that the proposed protocol is an effective pathway to achieve the desired functionalized surfaces. Copyright © 2010 John Wiley & Sons, Ltd.     

Two Routes for Immobilization of a Hyperbranched OH-Terminated Polyester on a Silicon Surface

Summary: Stable thin films of a hyperbranched aromatic-aliphatic OH-terminated polyester (POH) were prepared on silicon substrates. We report on grafting-to processes using macromolecular (PGMA) and bifunctional low-molecular weight coupling agents (BOX). An appropriate annealing procedure was developed for the covalent immobilization. The HBP films were characterized with spectroscopic ellipsometry, drop shape analysis (DSA), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and electrokinetic measurements. The immobilized films have an uniform surface morphology and are slightly hydrophilic (θ_a = 79°). The grafting process did not influence the chemical composition and the surface properties, which is important for the further application as functional layers in aqueous media.     

“电极/溶液”界面的椭圆偏振测量技术研究进展

椭圆偏振测量技术是通过解析偏振光束在界面上或薄膜中反射或透射时偏振状态的变化,获取界面或薄膜的厚度、复折射率等性质的一种光学方法,是一种高灵敏度、非破坏性的原位实时表征技术,被广泛应用于“电极/溶液”界面的研究. 本文简要介绍了椭圆偏振测量技术的基本原理及其最新发展,并着重评述了能源电化学、材料电化学、电分析与生物电化学等领域中,应用椭圆偏振测量技术研究“电极/溶液”界面的现状.     

Structure of Protein Layers during Competitive Adsorption

The formation of protein layers during competitive adsorption was studied with ellipsometry. Single, binary, and ternary protein solutions of human serum albumin (HSA), IgG, and fibrinogen (Fgn) were investigated at concentrations corresponding to blood plasma diluted 1/100. As a model surface, hydrophobic hexamethyldisiloxane (HMDSO) plasma polymer modified silica was used. By using multiambient media measurements of the bare substrate prior to protein adsorption the adsorbed amount as well as the thickness and refractive index of the adsorbed protein layer could be followedin situand in real time. Under conditions used in these experiments neither IgG nor fibrinogen could fully displace serum albumin from the interface. The buildup of the protein layer occurred via different mechanisms for the different protein systems. Fgn adsorbed in a rather flat orientation at low adsorbed amounts, while at higher surface coverage the protein reoriented to a more upright orientation in order to accommodate more molecules in the adsorbed layer. IgG adsorption proceeded mainly end-on with little reorientation or conformational change on adsorption. Finally, for HSA an adsorbed layer thickness greater than the molecular dimensions was observed at high concentrations (although not at low), indicating that aggregates or multilayers formed on HMDSO plasma polymer surfaces. For all protein mixtures the adsorbed layer structure and buildup indicated that Fgn was the protein dominating the adsorbed layer, although HSA partially blocked the adsorption of this protein. At high surface concentration, HSA/Fgn mixtures show an abrupt change in both adsorbed layer thickness and refractive index suggesting, e.g., an interfacial phase transition of the mixed protein layer. A similar but less pronounced behavior was observed for HSA/IgG. For IgG/Fgn and HSA/IgG/Fgn a buildup of the adsorbed layer similar to that displayed by Fgn alone was observed.     

Evaluation of different nonspecific binding blocking agents deposited inside poly(methyl methacrylate) microfluidic flow-cells

Poly(methyl methacrylate) (PMMA) flow-cells containing microwells were deposited with different nonspecific binding blocking agents, namely, bovine serum albumin (BSA), cationic lipid (DOTAP:DOPE) and diethylene glycol dimethyl ether (DEGDME). Water contact angle (WCA) and atomic force microscope (AFM) measurements were carried out to confirm the successful depositions of BSA, DOTAP, and DEGDME onto the PMMA surfaces. Fluorescent intensity measurements were performed to evaluate the degree of nonspecific adsorption of Cy5-labeled anti-IgG proteins onto plain and oxygen plasma-treated (PT) PMMA flow-cells as well as PMMA flow-cells deposited with different above-mentioned blocking agents. We then employed a label-free detection method called total internal reflection ellipsometry (TIRE) to evaluate the stability of the deposited blocking agents inside the PMMA flow-cells. It was found that, while DOTAP:DOPE was the best agent for blocking the nonspecific adsorption, it could be removed from the PMMA surfaces of the flow-cells upon rinsing with phosphate buffered saline (PBS) and later deposited back onto the Au-coated glass sensing substrate of the TIRE. The removal of the blocking agents from PMMA surfaces and their deposition onto the sensing substrate were further manifested by measuring the kinetics and the amount of adsorbed anti-α-hCG proteins. Overall, the dry DEGDME coating by plasma-enhanced chemical vapor deposition (PECVD) showed very good blocking and excellent stability for subsequent assay inside the microwells. Our results could be useful when one considers what blocking agents should be used for PMMA-based microfluidic immunosensor or biosensor devices by looking at both the blocking efficiency and the stability of the blocking agent.     

Preparation and characterization of thin films of SiO(x) on gold substrates for surface plasmon resonance studies

We report here on the fabrication and characterization of stable thin films of amorphous silica (SiO(x)) deposited on glass slides coated with a 5 nm adhesion layer of titanium and 50 nm of gold, using the plasma-enhanced chemical vapor deposition (PECVD) technique. The resulting surfaces were characterized using atomic force microscopy (AFM), ellipsometry, contact angle measurements, and surface plasmon resonance (SPR). AFM analysis indicates that homogeneous films of silica with low roughness were formed on the gold surface. The deposited silica films showed excellent stability in different solvents and in piranha solution. There was no significant variation in the thickness or in the SPR signal after these harsh treatments. The Au/SiO(x) interfaces were investigated for their potential applications as new surface plasmon resonance sensor chips. Silica films with thicknesses up to 40 nm allowed visualization of the surface plasmon effect, while thicker films resulted in the loss of the SPR characteristics. SPR allowed further the determination of the silica thickness and was compared to ellipsometric results. Chemical treatment of the SiO(x) film with piranha solution led to the generation of silanol surface groups that have been coupled with a trichlorosilane.     

Plasmon microscopy and imaging ellipsometry of Artrobacter oxydans attached on polymer films

The attachment of Artrobacter oxydans 1,388 on a newly synthesized biodegradable copolymer of poly-(hexanlactam)-co-block-poly-(delta-valerolactone) is investigated by optical, microscopic and biochemical methods. The potentials of surface plasmon microscopy and imaging ellipsometry for detecting microorganisms when Al films are used to excite plasmons is assessed by comparing images obtained by these methods with dark field microscopy pictures. The experimental results demonstrate that in this case imaging ellipsometry and plasmon microscopy in transmission are promising methods and can be used in optical sensors for monitoring cell adhesion.     

掠射椭圆偏振术对K4[Fe(CN)6]/K3[Fe(CN)6]表面溶液层性质的研究

目前广泛应用于表面反应现场研究的椭圆偏振技术都采用反射式测量方案[1，2]，这种方法通过测量光在电极表面反射时描述偏振光相位变化的参量和振幅变化的参量．以及上述两个参量的变化趋势并结合预先设定的表面膜模型计算表面膜厚度和复折射系数等光学参量[3]反射式椭圆偏振测     

A novel Surface Plasmon Resonance enhanced Total Internal Reflection Ellipsometric application: electrochemically grafted isophthalic acid nanofilm on gold surface

The scope of this study is to modify a Surface Plasmon Resonance (SPR) sensor slide with isophthalic acid to evaluate the possible application on the detection of copper(II) ions in aqueous media by total internal reflection ellipsometry. A gold sensor surface was modified by an electrochemical diazonium reduction modification method. The modified surfaces are characterized with cyclic voltammetry (CV) and ellipsometry. Isophthalic acid monolayer modified gold slides were used for in situ detection of aqueous Cu(2+) solution with the SPR enhanced total internal reflection ellipsometry (SPRe-TIRE) technique. Layer formation, pH dependency of adsorption, sensor response of the SPRe-TIRE and isothermal kinetic parameters were examined. A high dependency on the number of CV cycles in the monolayer-multiple layer transition was observed. The suggested sensor gave a linear response over a wide range of Cu(2+) concentrations. It was also reported that adsorption on the SPRe-TIRE sensor gave Langmuir adsorption model behavior.     

Vacuum ultraviolet optical analysis of spin‐cast chitosan films modified by succinic anhydride and glycidyl phenyl ether

Optical properties of spin‐cast chitosan films were determined in the vacuum ultraviolet (VUV) through visible regions of the spectrum using spectroscopic ellipsometry. The onset of absorption in the ultraviolet was determined for chitosan films modified with succinic anhydride (SA) and glycidyl phenyl ether (GPE). This absorption was accounted for by including Gaussian and Tauc–Lorentz shaped oscillators in the optical model. VUV through visible optical constants were determined for these modified films, as well as for immunoglobulin G (IgG) attached to GPE‐modified samples. Several resonant oscillator structures exhibit greatly differing oscillator parameters for the as‐deposited, as well as SA‐ and GPE‐modified films. These in‐plane and out‐of‐plane oscillator strengths, energy positions, and broadening were determined and tabulated, and their chemical origins identified. A phenyl resonant π‐bond at approximately 6.3 eV was observed for the GPE‐modified sample. This was not present in the bare chitosan data. GPE contains a phenyl group in the molecule and absorption due to it is clearly identified. Clarity of data after surface chemical modification is due to the high surface sensitivity of VUV ellipsometry. Copyright © 2007 John Wiley & Sons, Ltd.     

Quantitative chemical composition of thin films with infrared spectroscopic ellipsometry: application to hydrated oxide films on aluminium

A method to quantify the composition of thin films using infrared spectroscopic ellipsometry (IRSE), supplemented by visible spectroscopic ellipsometry (VISSE), is proposed. Because ellipsometry measures the thickness and optical constants of a surface layer simultaneously, the absorption coefficient of the film as a function of wavelength can be obtained. Using values of the absorption coefficients for the pure components of the film, the percentages (mol.% or wt.%) of each component in the film can be calculated. The method is demonstrated in a study of the hydration of oxide films on electropolished aluminium and the anodically formed barrier oxide film. The IRSE technique shows that hydration of the films by immersion in boiling water results in the conversion of aluminium oxide to pseudoboehmite. Copyright © 2003 John Wiley & Sons, Ltd.     

Comparative thickness measurements of SiO2/Si films for thicknesses less than 10 nm

We report on a comparative measurement of SiO₂/Si dielectric film thickness (t < 10 nm) using grazing‐incidence x‐ray photoelectron spectroscopy, neutron reflectometry and spectroscopic ellipsometry. Samples with nominal thicknesses of 3–7 nm were characterized by XPS with grazing‐incidence x‐rays at 1.8 keV, by cold neutron reflectometry (λ = 0.475 nm) and by spectroscopic ellipsometry over 1.5 eV < E < 6.0 eV. The results show good agreement between the ellipsometry and grazing‐incidence XPS, with slightly lower values for the neutron reflectometry. The role of surface contamination in each type of measurement is discussed. Published in 2004 by John Wiley & Sons, Ltd.     

Surface characterization of functional poly(diacetylene) and poly(butadiene) mono- and multilayers

The surface properties of Langmuir-Blodgett mono- and multilayers of a variety of amphiphilic poly(diacetylene)s and poly(butadiene)s were investigated by contact angle, streaming potential, ellipsometry, and X-ray photoelectron spectroscopic (XPS) measurements. Captive air and octane angles varied between approximately 60° and 105° for hydrophobicx-layers and 31° to 46° for hydrophilic surfaces depending on the particular head group, whereas advancing angles determined via the vertical plate method are considerably higher. Negative streaming potentials were obtained for all surfaces. Positively charged monolayers yielded less negative- potential values (–28 mV) than negatively charged (–52 mV) or hydrophobic (–50 mV) layers. Ellipsometry measurements yielded an average layer thickness of 27±6 Å for 3 to 11 layers. X-ray photoelectron spectroscopy results qualitatively confirmed the expected composition. All of the samples, which were handled and stored in air after deposition and polymerization, were surface oxidized.Dedicated to Prof. Dr. F. H. Müller.     

Towards understanding of protein adsorption behavior on plasma polymerized pyrrole film

Plasma polymerized pyrrole-like (PPpy) films exhibit good environmental stability and offer high reactivity with biomolecules. The present paper follows on from previous work on PPpy films applied as DNA immobilization/hybridization and describes the adsorption kinetics of bovine serum albumin (BSA) on PPpy films. Atom force microscopy was used to detect the surface roughness of PPpy surfaces obtained at different input powers or for different polymerization time, including the surface roughness before and after BSA adsorption. The influence of experimental conditions (i.e., the plasma input power, the polymerization time, the concentration of BSA, and the pH values of buffer solutions) on protein adsorption was investigated in situ by Surface plasmon resonance spectroscopy (SPR). SPR analysis confirmed the differently dynamic adsorption behavior of BSA on PPpy films under various experimental conditions. The adsorption constant, K _a , was deduced from Langmuir isotherm equations, which were simulated using experimental data collected by SPR and electrochemical impedance spectroscopy (EIS). Analysis of the combination data of SPR and EIS indicates that PPpy films under various conditions show completely different adsorption behaviors and could be applied as biomaterials for electrochemical protein sensing or as protein-resistant.      

椭圆偏振光谱方法对电化学的研究及应用

椭圆偏振光在固体表面入射和反射后,光学参量△,Ψ及其所表征的偏振状态,将因固体表面膜的厚度、性质不同而发生相应的变化．测定△,Ψ及其变化规律、可为固体材料、表面膜厚度、表面或界面上发生的有关物理或化学变化等直接或间接地提供信息,其灵敏度可反映表面膜层０．１～０．０１ｎｍ厚度的变化．因此,椭圆偏振光谱（下称椭圆法）在固体物理、表面化学、金属表面腐蚀、生物大分子表面等研究领域具有重要价值［１］．该法能检测的表面膜厚相当于电极上的单分子层的尺寸,特别适用于从分子水平上研究电化学体系,是最灵敏的光谱电化…     

金属锌表面膜形成的椭圆偏振技术研究

介绍了近年作者课题组使用椭圆偏振技术研究金属锌表面氧化膜的形成,包括多晶锌表面自然氧化物薄膜的形成及其光学性能和电子结构、不同气氛自然氧化物膜的生长研究以及在碱性碳酸盐介质金属锌的电化学过程等方面的工作. 旨在通过原位和非原位椭圆偏振技术了解金属锌表面氧化物膜层的光、电性能以及膜层结构的改变和生长动力学,这对评估锌氧化层的总体性能有着重要意义.     

A microarray chip for label-free detection of narcotics

A protein array chip for label-free optical detection of low molecular weight compounds has been developed. As a proof of principle, the chip is proven capable of rapidly (approximately 1 min) determining hits from aqueous cocktails composed of four common narcotics, cocaine, ecstasy, heroin, and amphetamine, using imaging surface plasmon resonance (SPR) as the detection principle. The chip is produced by injecting a mixture of antibodies and letting them self-sort and bind to narcotic analog coupled proteins already present in a predefined pattern on the supporting substrate. An indirect detection method, where antibodies are displaced from the surface upon recognition of their corresponding narcotics, is used to obtain the optical contrast and thus a detectable SPR and/or ellipsometric signal. Two types of readouts are possible from the present setup: intensity SPR images and SPR/ellipsometric sensorgrams. Positive hits were routinely obtained for analyte concentrations of 50 pg/μL and the limit of detection, without any parameter optimizations, seems to fall in the range 0.5 pg/μL (1.4 nM) for heroin, 2.5 pg/μL (8.2 nM) for cocaine, and 5 pg/μL for the other two narcotics (26 nM for ecstasy and 37 nM for amphetamine). With improved readout possibilities (sampling frequency), signal evaluation algorithms, and antibody–antigen design strategies, we believe this limit can be further improved. The chip is shown to work for many measurement cycles with excellent reproducibility. Moreover, with a more advanced fluidic system, excess injected antibodies could be collected and reused for many cycles, which could make the running costs of the system very low. The chip is in no way limited to detection of narcotics. Other low molecular weight compounds could easily be detected on the same chip. For example, trinitrotoluene detection has already been demonstrated using our chip. Possible areas of application for the system are therefore envisaged in airport and underground transport security, customs, drug interdiction, forensics, and as warning alerts on military equipment and personnel. Figure Narcotics chip (left) composed of spots of piezodispensed analog-coupled proteins that are loaded with antibodies to form a patterned regions represented by the capital letter of the four different narcotics in focus. (Right) The same chip showing hits for ectasy and herion in the cocktail. Both images are obtained in imaging surface plasmon resonance mode