Characterization of phospholipid bilayer formation on a thin film of porous SiO2 by reflective interferometric Fourier transform spectroscopy (RIFTS)

Classical methods for characterizing supported artificial phospholipid bilayers include imaging techniques such as atomic force microscopy and fluorescence microscopy. The use in the past decade of surface-sensitive methods such as surface plasmon resonance and ellipsometry, and acoustic sensors such as the quartz crystal microbalance, coupled to the imaging methods, have expanded our understanding of the formation mechanisms of phospholipid bilayers. In the present work, reflective interferometric Fourier transform spectrocopy (RIFTS) is employed to monitor the formation of a planar phospholipid bilayer on an oxidized mesoporous Si (pSiO(2)) thin film. The pSiO(2) substrates are prepared as thin films (3 μm thick) with pore dimensions of a few nanometers in diameter by the electrochemical etching of crystalline silicon, and they are passivated with a thin thermal oxide layer. A thin film of mica is used as a control. Interferometric optical measurements are used to quantify the behavior of the phospholipids at the internal (pores) and external surfaces of the substrates. The optical measurements indicate that vesicles initially adsorb to the pSiO(2) surface as a monolayer, followed by vesicle fusion and conversion to a surface-adsorbed lipid bilayer. The timescale of the process is consistent with prior measurements of vesicle fusion onto mica surfaces. Reflectance spectra calculated using a simple double-layer Fabry-Perot interference model verify the experimental results. The method provides a simple, real-time, nondestructive approach to characterizing the growth and evolution of lipid vesicle layers on the surface of an optical thin film.     

Optical technologies for the read out and quality control of DNA and protein microarrays

Microarray formats have become an important tool for parallel (or multiplexed) monitoring of biomolecular interactions. Surface-immobilized probes like oligonucleotides, cDNA, proteins, or antibodies can be used for the screening of their complementary targets, covering different applications like gene or protein expression profiling, analysis of point mutations, or immunodiagnostics. Numerous reviews have appeared on this topic in recent years, documenting the intriguing progress of these miniaturized assay formats. Most of them highlight all aspects of microarray preparation, surface chemistry, and patterning, and try to give a systematic survey of the different kinds of applications of this new technique. This review places the emphasis on optical technologies for microarray analysis. As the fluorescent read out of microarrays is dominating the field, this topic will be the focus of the review. Basic principles of labeling and signal amplification techniques will be introduced. Recent developments in total internal reflection fluorescence, resonance energy transfer assays, and time-resolved imaging are addressed, as well as non-fluorescent imaging methods. Finally, some label-free detection modes are discussed, such as surface plasmon microscopy or ellipsometry, since these are particularly interesting for microarray development and quality control purposes.     

Au nanoparticle monolayers covered with sol-gel oxide thin films: optical and morphological study

In this work, we provide a detailed study of the influence of thermal annealing on submonolayer Au nanoparticle deposited on functionalized surfaces as standalone films and those that are coated with sol-gel NiO and TiO(2) thin films. The systems are characterized through the use of UV-vis absorption, X-ray diffraction (XRD), atomic force microscopy (AFM), scanning electron microscopy (SEM), and spectroscopic ellipsometry. The surface plasmon resonance peak of the Au nanoparticles was found to red-shift and increase in intensity with increasing surface coverage, an observation that is directly correlated to the complex refractive index properties of Au nanoparticle layers. The standalone Au nanoparticles sinter at 200 °C, and a relationship between the optical properties and the annealing temperature is presented. When overcoated with sol-gel metal oxide films (NiO, TiO(2)), the optical properties of the Au nanoparticles are strongly affected by the metal oxide, resulting in an intense red shift and broadening of the plasmon band; moreover, the temperature-driven sintering is strongly limited by the metal oxide layer. Optical sensing tests for ethanol vapor are presented as one possible application, showing reversible sensing dynamics and confirming the effect of Au nanoparticles in increasing the sensitivity and in providing a wavelength dependent response, thus confirming the potential use of such materials as optical probes.     

New approach to writing and simultaneous reading of micropatterns: combining surface plasmon resonance imaging with scanning electrochemical microscopy (SECM)

This work establishes the compatibility of surface plasmon resonance imaging (SPR-i) with the visualization of localized electropolymerization. The "writing" of polypyrrole and polypyrrole-oligonucleotide patterns onto thin gold films is demonstrated using scanning electrochemical microcopy (SECM), while an optical method, SPR-i, simultaneously detected the formed micropatterns. The combination of these two methods, SECM/SPR-i, has the advantage of not only controlling the patterning process but also providing unique information on the micropattern formation. The influence of the pulsing time and the monomer concentration on the spot size and its characteristics has been investigated in detail using SPR-i. Fluorescence microscopy and atomic force microscopy have also been used to support the data obtained by SPR-i.     

Total Internal Reflection Ellipsometry under SPR Conditions: In-Situ Monitoring of the Growth of Poly(N-isopropylacrylamide) (PNIPAAm) Brushes

Total internal reflection ellipsometry (TIRE) under surface plasmon resonance (SPR) conditions represents a powerful characterization technique combining the conveniences of spectroscopic ellipsometry with SPR. Besides the very high sensitivity to small changes in the optical constants (up to 10 times more sensitive than conventional ellipsometry), the possibility to investigate media of different optical densities or even opaque media makes this analytical method very convenient for different sensing applications. This article presents an example of application of TIRE under SPR conditions for the continuously in-situ monitoring of the growth of covalently tethered poly(N-isopropylacrylamide) (PNIPAAm) chains on a gold surface.     

Near-field optical imaging of enhanced electric fields and plasmon waves in metal nanostructures

In this article, studies on noble metal nanostructures using near-field optical microscopic imaging are reviewed. We show that near-field transmission imaging and near-field two-photon excitation imaging provide valuable methods for investigation of plasmon resonances in metal nanostructures. The eigenfunctions of plasmon modes in metal nanoparticles are directly visualized using these methods. For metal nanowire systems, wavevectors of the longitudinal plasmon modes can be estimated directly from the wave-function images, and the dispersion relations are plotted and analyzed. Using ultrafast transient near-field imaging, we show that the deformation of the plasmon wave function takes place after photoexcitation of a gold nanorod. Such methods of plasmon-wave imaging may provide a unique basic tool for designing plasmon-mode-based nano-optical devices. We also demonstrate that the near-field two-photon excitation probability images reflect localized electric-field enhancements in metal nanostructures. We apply this method to gold nanosphere assemblies and clearly visualize the local enhanced optical fields in the interstitial sites between particles (hot spots). We also show the contribution of hot spots to surface enhanced Raman scattering. The methodology described here may provide valuable basic information about the characteristic enhanced optical fields in metal nanostructures as well as on their applications to new innovative research areas beyond the conventional scope of materials.     

Gold Nanorods as Contrast Agents for Biological Imaging: Optical Properties,Surface Conjugation and Photothermal Effects†

Gold nanorods (NRs) have plasmon‐resonant absorption and scattering in the near‐infrared (NIR) region, making them attractive probes for in vitro and in vivo imaging. In the cellular environment, NRs can provide scattering contrast for darkfield microscopy, or emit a strong two‐photon luminescence due to plasmon‐enhanced two‐photon absorption. NRs have also been employed in biomedical imaging modalities such as optical coherence tomography or photoacoustic tomography. Careful control over surface chemistry enhances the capacity of NRs as biological imaging agents by enabling cell‐specific targeting, and by increasing their dispersion stability and circulation lifetimes. NRs can also efficiently convert optical energy into heat, and inflict localized damage to tumor cells. Laser‐induced heating of NRs can disrupt cell membrane integrity and homeostasis, resulting in Ca²⁺ influx and the depolymerization of the intracellular actin network. The combination of plasmon‐resonant optical properties, intense local photothermal effects and robust surface chemistry render gold NRs as promising theragnostic agents.     

Proteomic applications of surface plasmon resonance biosensors: analysis of protein arrays

Proteomics is one of the most important issues in the post-genomic area, because it can greatly contribute to identifying protein biomarkers for disease diagnosis and drug screening. Protein array is a key technology for proteome researches and has been analyzed by various methods including fluorescence, mass spectrometry, atomic force microscopy and surface plasmon resonance (SPR). SPR biosensor is a promising technology in proteomics, since it has various advantages including real-time measurement of biomolecular interactions without labeling and the simple optical system for the device. SPR biosensors have a strong potential for analyzing proteomes by SPR imaging and SPR spectroscopic imaging, even though the challenge is to produce proteins on a proteomic scale.     

Newly developed chemical probes and nano-devices for cellular analysis.

A cellular analyzing system including a "real-time cellular imaging system" and a "comprehensive analyzing system for cellular responses" was developed. A "real-time cellular imaging system" is a system used to measure real-time imaging of multiple phenomena of a single cell with high special and temporal resolutions for the purpose to understand the pathology and physiology in a single cell and realize to single cell level diagnosis. A "real-time cellular imaging system" includes multi-probe imaging with AFM (atomic force microscopy), optical and SECM (scanning electrochemical microscopy) modes, which provides us with topological information and biochemical reactions at the local area of the interior and exterior of a cell. Scanning electrochemical/optical microscopy was applied to image PC12 cells. On the other hand, cells respond to their specific substances via their ligands. Therefore, the comprehensive analysis of protein-protein interaction is the important issue to determine the functions of cells. For this purpose, a "comprehensive analysis system for cellular responses" was developed. This system is based on SPR (surface plasmon resonance) and MS (mass spectrometry) using a nano-fabricated substrate. The interaction between IL-1 beta and anti-IL-1 beta antibodies was detected.     

Monolayers of polyethilenimine on flat glass: a versatile platform for cations coordination and nanoparticles grafting in the preparation of antibacterial surfaces

A polyethylenimine (PEI) self-assembled monolayer (SAM) is prepared, capable of complexing silver and copper cations and of anchoring silver nanoparticles, exerting antibacterial activity against Escherichia coli and Staphylococcus aureus. Functionalized glassy surfaces have been fully characterized through spectroscopic techniques (UV-Vis spectroscopy, spectroscopic ellipsometry), atomic force microscopy imaging and quantitative Ag and Cu analysis (ICP optical emission spectroscopy).     

Nanoclusters in polymer matrices prepared by co-deposition from a gas phase

This paper reviews the fabrication of organic and metal nanoclusters in polymer matrices by three co-deposition techniques. In particular, the structure and properties of polytetrafluoroethylene (PTFE), polychlortrifluoroethylene (PCTFE), polyparaphenylene sulphide (PPS), polystyrene (PS) and polyparaxylylene (PPX) films, containing gold (Au) and dye clusters are discussed. For the first time, dye-filled polymers and multi-component films, consisting of both Au nanoparticles and dye molecules, dispersed in the PTFE matrix were studied. A low temperature plasma was used for film structure modification. Cluster formation process was studied using optical spectroscopy in situ. Transmission electron microscopy (TEM), atomic force microscopy (AFM) and ellipsometry were used for characterisation of the grown films. During Au-PTFE film growth plasmon band shifted from 460-480 nm to 560 nm. Au cluster diameter was in the 3-7 nm range. Plasma treatment of the vapours led to formation of smaller, but more aggregated clusters. During Au-PPS film deposition a two-step growth mechanism was discovered. At the beginning of film growth the plasmon band at 540 nm appeared, but as thickness increased, the band at 430 nm dominated. Without plasma treatment a disordered mixture was deposited, while with plasma treatment large Au aggregates confined with PPS matrix having plasmon band at 620 nm were formed. Dye cluster formation depends on the dye ability to aggregate, its concentration and the properties of the polymer matrix. But cluster formation can also be tuned by varying the deposition conditions. Laser beam evaporation promoted cluster formation, while plasma treatment and dilution in a polymer matrix prevented cluster formation. In all cases both equilibrium and non-equilibrium film structure can be formed using kinetic factor. Asymmetric molecules with bulky substituents were oriented in polymer matrices by applying an electric field in situ or by corona poling. These molecules did not aggregate even at high dye load. The films exhibited second harmonic generation, which demonstrated chromophore orientation in the polymer matrices.     

Dendrimer-functionalized self-assembled monolayers as a surface plasmon resonance sensor surface

We report here a multistep route for the immobilization of DNA and proteins on chemically modified gold substrates using fourth-generation NH(2)-terminated poly(amidoamine) dendrimers supported by an underlying amino undecanethiol (AUT) self-assembled monolayer (SAM). Bioactive ultrathin organic films were prepared via layer-by-layer self-assembly methods and characterized by fluorescence microscopy, variable angle spectroscopic ellipsometry, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and attenuated total internal reflection Fourier transform infrared spectroscopy (ATR-FTIR). The thickness of the AUT SAM base layer on the gold substrates was determined to be 1.3 nm from ellipsometry. Fluorescence microscopy and AFM measurements, in combination with analyses of the XPS/ATR-FTIR spectra, confirmed the presence of the dendrimer/biopolymer molecules on the multilayer sensor surfaces. Model proteins, including streptavidin and rabbit immunoglobulin proteins, were covalently attached to the dendrimer layer using linear cross-linking reagents. Through surface plasmon resonance measurements, we found that sensor surfaces containing a dendrimer layer displayed an increased protein immobilization capacity, compared to AUT SAM sensor surfaces without dendrimer molecules. Other SPR studies also revealed that the dendrimer-based surfaces are useful for the sensitive and specific detection of DNA-DNA interactions. Significantly, the multicomponent films displayed a high level of stability during repeated regeneration and hybridization cycles, and the kinetics of the DNA-DNA hybridization process did not appear to be influenced by surface mass transport limiting effects.     

Chemical sensing and imaging with metallic nanorods

In this Feature Article, we examine recent advances in chemical analyte detection and optical imaging applications using gold and silver nanoparticles, with a primary focus on our own work. Noble metal nanoparticles have exciting physical and chemical properties that are entirely different from the bulk. For chemical sensing and imaging, the optical properties of metallic nanoparticles provide a wide range of opportunities, all of which ultimately arise from the collective oscillations of conduction band electrons ("plasmons") in response to external electromagnetic radiation. Nanorods have multiple plasmon bands compared to nanospheres. We identify four optical sensing and imaging modalities for metallic nanoparticles: (1) aggregation-dependent shifts in plasmon frequency; (2) local refractive index-dependent shifts in plasmon frequency; (3) inelastic (surface-enhanced Raman) light scattering; and (4) elastic (Rayleigh) light scattering. The surface chemistry of the nanoparticles must be tunable to create chemical specificity, and is a key requirement for successful sensing and imaging platforms.     

Ellipsometric identification of collective optical properties of silver nanocrystal arrays

The optical properties of silver nanocrystal arrays are investigated using spectroscopic ellipsometry in combination with polarized reflection measurements. Analysis of the ellipsometry and reflectometry spectra in terms of the "thin island film" theory enables a transparent identification of the contribution of collective effects to the optical response. Negligible image charge effects imply that only dipole contributions have to be considered. The interactions between the hexagonally ordered silver nanocrystals give rise to an effective modification of the spherical response to oblate entities with different polarizabilities parallel and perpendicular to the substrate, expressed in terms of corresponding depolarization factors. The effect of nanocrystal ordering, nearest-neighbor distance, size distribution, surrounding ambient, and the optical properties of the single nanocrystals on the optical response are analyzed. The extent of plasmon resonance peak splitting as a function of surface coverage is discussed.     

免标记光学生物传感器研究进展

免标记光学生物传感器因分析样品无需标记、强特异性、动态测量、无损检测、分析速度快等诸多优点,在化学、药学及生物等诸多领域中得到了快速发展和广泛应用。本文重点介绍了当前发展成熟且具代表性的四种免标记光学生物传感器,即表面等离子体共振传感器、光波导光模光谱传感器、椭圆偏振光学传感器以及反射干涉传感器,对每种传感器的原理结构、方法发展及在生化分析等相关领域中的最新应用进行了总结和论述;在此基础上,对它们各自的性能进行了优劣比较;最后对免标记光学生物传感器的发展前景进行了展望。     

Protein Microarrays on Carboxymethylated Dextran Hydrogels: Immobilization, Characterization and Application

Tetraoctadecylammonium bromide (TOAB, (CH₃(CH₂)₁₇)₄N⁺Br^–) has been used to print temporary hydrophobic barriers on carboxymethylated dextran (CMD) hydrogels to create a generic platform for protein microarray applications. The primary reason for printing temporary hydrophobic barriers is to prevent cross-contamination and overflow during microdrop dispensing. Equally important is to eliminate the risk for non-specific binding to the barriers during analyte exposure. This has been accomplished by introducing a regeneration step that removes the barriers after ligand immobilization. The overall fabrication process was characterized by microscopic wetting, atomic force microscopy, imaging ellipsometry, fluorescence microscopy, surface plasmon microscopy and biospecific interaction analysis. A series of model proteins including transferrin, Protein A, anti-myoglobin and bovine serum albumin was spotted into the TOAB-defined areas under different experimental conditions, e.g. at increased humidity and reduced substrate temperature or with glycerol as an additive in the protein solution. Much emphasis was devoted to studies aiming at exploring the homogeneity and activity of the immobilized proteins. The printed barriers were removed after protein immobilization using tert-n-butyl alcohol (TBA). TBA was found to be a very efficient agent as compared to previously used salt regeneration solutions, and the regeneration time could be reduced from 30 to 10 minutes. Finally, the potential of using the well established CMD hydrogel chemistry as a platform for protein microarrays was exploited using surface plasmon microscopy.     

Surface modification of confined microgeometries via vapor-deposited polymer coatings

The development of generally applicable protocols for the surface modification of complex substrates has emerged as one of the key challenges in biotechnology. The use of vapor-deposited polymer coatings may provide an appealing alternative to the currently employed arsenal of surface modification methods consisting mainly of wet-chemical approaches. Herein, we demonstrate the usefulness of chemical vapor deposition polymerization for surface modification in confined microgeometries with both nonfunctionalized and functionalized poly(p-xylylenes). For a diverse group of polymer coatings, homogeneous surface coverage of different microgeometries featuring aspect ratios as high as 37 has been demonstrated based on optical microscopy and imaging X-ray photoelectron spectroscopy. In addition, height profiles of deposited polymer footprints were obtained by atomic force microscopy and imaging ellipsometry indicating continuous transport and deposition throughout the entire microchannels. Finally, the ability of reactive coatings to support chemical binding of biological ligands, when deposited in previously assembled microchannels, is demonstrated, verifying the usefulness of the CVD coatings for applications in micro/nanofluidics, where surface modifications with stable and designable biointerfaces are essential. The fact that reactive coatings can be deposited within confined microenvironments exhibits an important step toward new device architectures with potential relevance to bioanalytical, medical, or "BioMEMS" applications.     

表面等离子体共振光谱技术与电化学方法联用及其应用

表面等离子体共振（Surface Plasmon Resonance,SPR）技术是利用金属薄膜光学耦合产生的物理光学现象建立的一种非常灵敏的光学分析手段. 近年发展的电化学表面等离子体共振（Electrochemical Surface Plasmon Resonance,EC-SPR）是将时间分辨表面等离子体共振光谱技术与电化学方法联用的一种新技术. 本文介绍了SPR和EC-SPR的基本原理,并重点阐述了时间分辨SPR光谱技术与电化学方法联用及应用,该技术已广泛地应用于反应动态过程研究、生物化学传感器、电极/溶液界面的表征、动力学常数的测定以及生物分子相互作用等领域.     

Enhancement of Nonlinear Optical Scattering by Gold Nanoparticles through Aggregation-Induced Plasmon Coupling in the Near-Infrared

Gold nanoparticles (AuNPs) are regarded as promising building blocks in functional nanomaterials for sensing, drug delivery and catalysis. One remarkable property of these particles is the localized surface plasmon resonance (LSPR), which gives rise to augmented optical properties through local field enhancement. LSPR also influences the nonlinear optical properties of metal NPs (MNPs) making them potentially interesting candidates for fast, high resolution nonlinear optical imaging. In this work we characterize and discuss the wavelength dependence of the hyper-Rayleigh scattering (HRS) behavior of spherical gold nanoparticles (GNP) and gold nanorods (GNR) in solution, from 850 nm up to 1300 nm, covering the near-infrared (NIR) window relevant for deep tissue imaging. The high-resolution spectral data allows discriminating between HRS and two photon photoluminescence contributions. Upon particle aggregation, we measured very large enhancements (ca. 10⁴) of the HRS intensity in the NIR, which is explained by considering aggregation-induced plasmon coupling effects and local field enhancement. These results indicate that purposely designed coupled nanostructures could prove advantageous for nonlinear optical imaging and biosensing applications.     

Refractive index of sparse layers of adsorbed gold nanoparticles

Measurements are presented of the effective complex refractive index of a layer of gold nanoparticles adsorbed to a silicon wafer at low coverages. The measurements were made by means of variable-angle ellipsometry, and correlated with nanoparticle coverage determined from atomic force microscope images. The analysis establishes the effective refractive index of a uniform layer whose thickness equals the nanoparticle diameter. A simple empirical relationship is obtained for real component of refractive index as a function of the fractional nanoparticle coverage regardless of the nanoparticle size. The imaginary component also follows a simple relationship but only up to a certain coverage, above which it increases rapidly. These relationships may be useful in other contexts such as chemical or biosensors in which the nanoparticle coverage could be inferred from optical measurements such as ellipsometry, surface plasmon resonance spectroscopy, reflectometry, or interferometry.