Surface Plasmon Spectroscopy Research Articles

Resonance Surface Plasmon Spectroscopy: Low Molecular Weight Substrate Binding to Cytochrome P450

A new detection mechanism has been developed for low molecular weight substrate binding to heme proteins based on resonance localized surface plasmon spectroscopy. Cytochrome P450 has strong electronic transitions in the visible wavelength region. Upon binding of a substrate molecule (e.g., camphor), the absorption band of cytochrome P450 shifts to shorter wavelength. The event of camphor binding to a nanoparticle surface modified with cytochrome P450 protein receptors is monitored using UV-vis spectroscopy. It is observed for the first time that the binding of the substrate molecules to the protein receptor induces a blue-shift in the localized surface plasmon resonance (LSPR) of the nanosensors. The coupling between the molecular resonance of the substrate-free and substrate-bound cytochrome P450 proteins and the nanoparticles' LSPR leads to a highly wavelength-dependent LSPR response. When the LSPR of the nanoparticles is located at a wavelength distant from the cytochrome P450 resonance, an average of approximately 19 nm red-shift is observed upon cytochrome P450 binding to the nanoparticles and a approximately 6 nm blue-shift is observed upon camphor binding However, this response is significantly amplified approximately 3 to 5 times when the LSPR of the nanoparticles is located at a slightly longer wavelength than the cytochrome P450 resonance, that is, a 66.2 nm red-shift upon cytochrome P450 binding and a 34.7 nm blue-shift upon camphor binding. This is the first example of the detection of small molecules binding to a protein modified nanoparticle surface on the basis of LSPR.

Measurement of antibody binding to protein immobilized on gold nanoparticles by localized surface plasmon spectroscopy

Antibody binding to bovine serum albumin (BSA) and human serum albumin (HSA) immobilized onto gold nanoparticles was studied by means of localized surface plasmon resonance (LSPR) spectroscopy. Amine-modified glass was prepared by self-assembly of amine-terminated silane on substrate, and gold (Au) nanoparticles were deposited on the amine-modified glass substrate. Au nanoparticles deposited on the glass surface were functionalized by BSA and HSA. BSA immobilization was confirmed by LSPR spectroscopy in conjunction with surface-enhanced Raman scattering spectroscopy. Then, LSPR response attributable to the binding of anti-BSA and anti-HSA to BSA- and HSA-functionalized Au nanoparticles, respectively, was examined. Anti-HSA at levels larger than approximately 10 nM could be detected by HSA-immobilized chips with LSPR optical response, which was saturated at concentrations greater than approximately 650 nM of anti-HSA.

Layer-by-layer self-assembled conducting polymer films at elevated pressure investigated by surface plasmon spectroscopy with electrochemistry

Polyaniline/sulfonated polyaniline (PANI/SPANI) multilayer films were fabricated using the layer-by-layer (LbL) technique. The electrochemical and optical properties of the film at elevated pressure were investigated by high-pressure surface plasmon spectroscopy combined with electrochemistry. Cyclic voltammograms of the PANI/SPANI films were performed at different hydrostatic pressure. It was found that the charge transfer currents decrease with elevated pressure. This indicates that the film becomes more compact with increasing hydrostatic pressure, which is confirmed by surface plasmon spectra, hinting at a substantial increase in the optical density.

Spectroscopy of surface plasmons in metal films with nanostructures

The direct measurement of local surface plasmon polariton spectra in micron-sized areas of metal films is implemented through the near-field imaging of two-dimensional plasmon interference patterns. The plasmons are generated at circular nanoslits. The period of their interference yields the plasmon wave vector, and imaging at different excitation wavelengths yields the plasmon dispersion. The dielectric function of the film, retrieved by fitting the measured dispersion curve, differs from published values, underlining the necessity for the in situ determination of the dielectric function of metal films intended for plasmonic circuits, which can be achieved within a few micron areas using our technique.

Influence of Cholesterol Composition on the Association of Serum Mannan-Binding Proteins with Mannosylated Liposomes

In our previous studies, serum mannan-binding protein (MBP) accelerated the uptake by cultured macrophages. The present study was initiated to investigate the kinetics of molecular interaction between mannosylated liposomes and MBP in more details and the effects of lipid composition on the interaction. The analysis was carried out by surface plasmon spectroscopy (SPR) methods, using rabbit serum MBP isolated by affinity chromatography. In SPR studies, neither conventional liposomes nor galactosylated liposomes indicated any interaction, but each mannosylated liposomes had a high response signal corresponding to molecular interaction with immobilized MBP. Association of mannosylated liposomes to serum MBP was not dependent on the lipid composition, suggesting a diffusion-controlled association. Dissociation of the mannosylated liposomes from serum MBP was extremely slow. DSPC/Chol/Man-C4-Chol (90 : 5 : 5, molar ratio) exhibited a slower dissociation rate than DSPC/Chol/Man-C4-Chol (60 : 35 : 5). Clustering of mannose residues on liposomal surfaces might be important in determining the binding affinity of mannosylated liposomes with MBP.

The Plant Plasma Membrane Ca2+ Pump ACA8 Contains Overlapping as Well as Physically Separated Autoinhibitory and Calmodulin-binding Domains

In plant Ca(2+) pumps belonging to the P(2B) subfamily of P-type ATPases, the N-terminal cytoplasmic domain is responsible for pump autoinhibition. Binding of calmodulin (CaM) to this region results in pump activation but the structural basis for CaM activation is still not clear. All residues in a putative CaM-binding domain (Arg(43) to Lys(68)) were mutagenized and the resulting recombinant proteins were studied with respect to CaM binding and the activation state. The results demonstrate that (i) the binding site for CaM is overlapping with the autoinhibitory region and (ii) the autoinhibitory region comprises significantly fewer residues than the CaM-binding region. In a helical wheel projection of the CaM-binding domain, residues involved in autoinhibition cluster on one side of the helix, which is proposed to interact with an intramolecular receptor site in the pump. Residues influencing CaM negatively are situated on the other face of the helix, likely to face the cytosol, whereas residues controlling CaM binding positively are scattered throughout. We propose that early CaM recognition is mediated by the cytosolic face and that CaM subsequently competes with the intramolecular autoinhibitor in binding to the other face of the helix.

Hydrogen Adsorption Isotherms: Modelling of Experimental Data

Adsorption isotherm measurements have shown deviations from classical theories in the past, leading to the inclusion of more complex effects into adsorption theory. We demonstrate here that in the case of hydrogen adsorption above the triple point measured by surface plasmon spectroscopy the main deviation is related to the measurement method itself. Modelling of the experimental data shows good agreement with the classical theoretical expectations, without the need of additional contributions.

Sterol Binding Assay Using Surface Plasmon Fluorescence Spectroscopy

We describe the design of a novel in vitro assay to study the interaction of soluble proteins with small hydrophobic sterol ligands. The sterol molecules are incorporated in an artificial membrane system in order to mimic their arrangement found in a biomembrane. The artificial membrane setup is monitored in real time by surface plasmon spectroscopy. Binding of fluorescently labeled soluble protein is observed by optical detection with surface plasmon enhanced fluorescence spectroscopy. By application of the novel assay, we demonstrate that four different oxidized sterol molecules are specifically recognized by the yeast protein Osh5p, a presumed oxysterol binding protein. Osh5p from yeast is the first oxysterol binding protein homologue for which oxysterol binding is shown with this new technique. With the design of our novel in vitro oxysterol binding assay, we have solved the technically challenging difficulty of presenting hydrophobic ligands to hydrophilic proteins in aqueous media.

Homopolymer and block copolymer brushes on gold by living anionic surface‐initiated polymerization in a polar solvent

AbstractLiving anionic surface‐initiated polymerization on flat gold substrates has been conducted to create uniform homopolymer and diblock copolymer brushes. A 1,1‐diphenylethylene (DPE) self‐assembled monolayer was used as the immobilized precursor initiator. n‐BuLi was used to activate the DPE in tetrahydrofuran at –78 °C to initiate the polymerization of different monomers (styrene, isoprene, ethylene oxide, and methyl methacrylate). Poly(styrene) (PS) and poly(ethylene oxide) (PEO) in particular were first investigated as grafted homopolymers, followed by their copolymers, including poly(isoprene)‐b‐poly(methylmethacrylate) (PI‐b‐PMMA). A combined approach of spectroscopic (Fourier transform infrared spectroscopy, surface plasmon spectroscopy, ellipsometry, X‐ray photoelectron spectroscopy) and microscopic (atomic force microscopy) surface analysis was used to investigate the formation of the polymer brushes in polar solvent media. The chemical nature of the outermost layer of these brushes was studied by water contact angle measurements. The effect of the experimental conditions (solvent, temperature, initiator concentration) on the surface properties of the polymer brushes was also investigated. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 769–782, 2006

Multi-analyte surface plasmon resonance biosensing

Surface plasmon resonance (SPR) biosensors are affinity sensing devices exploiting a special mode of electromagnetic field—surface plasmon-polariton—to detect the binding of analyte molecules from a liquid sample to biomolecular recognition elements immobilized on the surface of the sensor. In this paper, we review advances of SPR biosensor technology towards detection systems for the simultaneous detection of multiple analytes (multi-analyte detection). In addition, we report application of a recently developed multichannel SPR sensor based on spectroscopy of surface plasmons and wavelength division multiplexing of sensing channels to multi-analyte detection.

Multiple surface plasmon spectroscopy for study of biomolecular systems

We report a new approach to surface plasmon resonance (SPR) biosensing based on spectroscopy of multiple surface plasmons on a special multi-diffractive grating coupler. In this approach, the binding event at a surface of the SPR grating coupler is simultaneously observed by surface plasmons of different wavelengths, which makes it possible to determine binding-induced changes in the refractive index distribution at the sensor surface. We demonstrate the potential of this new concept in a model experiment in which three-surface plasmon spectroscopy is used to determine background refractive index variations and changes in the thickness and refractive index of a bovine serum albumin multi-layer grown on the surface of the SPR grating coupler.

Recognition of Phospholipids in Streptomyces Phospholipase D

To investigate the contribution of amino acid residues to the enzyme reaction of Streptomyces phospholipase D (PLD), we constructed a chimeric gene library between two highly homologous plds, which indicated different activity in transphosphatidylation, using RIBS (repeat-length independent and broad spectrum) in vivo DNA shuffling. By comparing the activities of chimeras, six candidate residues related to transphosphatidylation activity were shown. Based on the above result, we constructed several mutants to identify the key residues involved in the recognition of phospholipids. By kinetic analysis, we identified that Gly188 and Asp191 of PLD from Streptomyces septatus TH-2, which are not present in the highly conserved catalytic HXKXXXXD (HKD) motifs, are key amino acid residues related to the transphosphatidylation activity. To investigate the role of two residues in the recognition of phospholipids, the effects of these residues on binding to substrates were analyzed by surface plasmon spectroscopy. The result suggests that Gly188 and Asp191 are involved in the recognition of phospholipids in correlation with the N-terminal HKD motif. Furthermore, this study also provides experimental evidence that the N-terminal HKD motif contains the catalytic nucleophile, which attacks the phosphatidyl group of the substrate.

Electromagnetic Interactions in Plasmonic Nanoparticle Arrays

Single two-dimensional planar silver arrays and one-dimensional linear gold chains of nanoparticles were investigated by dark-field surface plasmon spectroscopy and studied as a function of interparticle distance, particle size, and number of particles. In agreement with recent theoretical predictions, a red shift of the surface plasmon resonance occurring in two-dimensional arrays was found for lattice spacings below 200 nm. This red shift is associated with a significant broadening of the resonance and is attributed to the onset of near-field interactions. We found that the relative contributions of the long-range and short-range interactions in two-dimensional arrays of particles are fundamentally different to those occurring in individual linear chains.

Salt softening of polyelectrolyte multilayer microcapsules

By using a combination of atomic force and confocal microscopy, we explore the effect of 1:1 electrolyte (NaCl) on the stiffness of polyelectrolyte microcapsules. We study the “hollow” and “filled” (with polystyrene sulfonate) capsules. In both cases the shells are composed of layers of alternating polystyrene sulfonate (PSS) and polyallylamine hydrochloride (PAH). The stiffness of both “hollow” and “filled” capsules was found to be largest in water. It decreases with salt concentration up to ∼3 mol/L and gets quasi-constant in more concentrated solutions. The “filled” capsules are always stiffer than “hollow.” The observed softening correlates with the salt-induced changes in morphology of the multilayer shells detected with the scanning electron microscopy. It is likely that at concentrations below ∼3 mol/L the multilayer shell is in a “tethered” state, so that the increase in salt concentration leads to a decrease in number of ionic cross-links and, as a result, in the stiffness. In contrast, above the critical concentration of ∼3 mol/L multilayer shells might be in a new, “melted,” state. Here the multilayer structure is still retained, but sufficient amount of ionic cross-links is broken, so that further increase in salt concentration does not change the capsule mechanics. These ideas are consistent with a moderate swelling of multilayers at concentrations below ∼3 mol/L and significant decrease in their thickness in more concentrated solutions measured with surface plasmon spectroscopy.

Rich information format surface plasmon resonance biosensor based on array of diffraction gratings

We report a new optical biosensor platform for high throughput label-free monitoring of biomolecular interactions. This biosensor platform is based on spectroscopy of surface plasmons on an array of miniature diffraction gratings. Each miniature grating serves as an independent sensing element and allows the monitoring of refractive index changes induced by biomolecular interactions on the grating surface. The presented sensor concept shows potential for real-time observation of thousands of interactions on a single sensor chip. In this paper, we demonstrate a prototype device consisting of an SPR sensor chip with 216 sensing channels, a fluidic system for sample distribution on the sensor chip and an optical chip readout system.

Laser spectroscopic investigation of a new precursor-type polyparaphenylenevinylene

Optical, structural and morphological properties of thin films of polyparaphenylenevinylene (PPV) formed by an alkyl sulfinyl precursor route have been studied. Thin films were fabricated on an optical glass and on quartz glass either by spin-coating of the precursor solution or by layer-by-layer deposition using Langmuir–Blodgett technique. PPV precursor films were also spin-coated on gold-coated glass in order to study thin-film optical parameters by surface plasmon spectroscopy. We have been successful in forming about 40 precursor mono layers on quartz glass by Langmuir–Blodgett technique using optimized surface pressure and dipping conditions. After thermal conversion of the precursor layers good quality fluorescent PPV films of yellow colour have been obtained. Optical characterization of the films was carried out by linear absorption and emission spectroscopy, ellipsometry, and surface plasmon spectroscopy. Structural and morphological studies on the thin films were carried out by using X-ray scattering and atomic force microscopy. Wave-guided travelling-wave laser action has been achieved in a PPV film on quartz glass. The sample was transversally pumped with picosecond laser pulses (wavelength 347.15 nm, duration 35 ps). Laser emission occurred at 550 nm for pump pulse energy densities above 1 × 10 - 4 J cm - 2 .

Incorporation of integrins into artificial planar lipid membranes: characterization by plasmon-enhanced fluorescence spectroscopy

An optimized peptide-tethered artificial lipid membrane system has been developed. Integrins (cell adhesion receptors) were functionally incorporated into this membrane model and integrin–ligand interactions were analyzed by surface plasmon-enhanced fluorescence spectroscopy (SPFS). The transmembrane receptors α vβ 3 and α 1β 1 of the integrin superfamily were incorporated into a lipid-functionalized peptide layer by vesicle spreading. Consecutive layer formations were monitored by surface plasmon spectroscopy (SPS). Orientation and accessibility of the membrane receptor α vβ 3 was reliably assessed by specific and reproducible binding of selective antibodies. Moreover, full retention of the functional properties of this receptor was verified by specific and reversible binding of natural ligands. Functional integrity of incorporated integrins was maintained over a time period of 72 h. The integrin/extracellular matrix ligand complexes, whose formations are known to depend on the presence of divalent cations, were lost upon addition of ethylenediaminetetraacetate. Therefore, regeneration of the surface for further binding experiments with minimized unspecific ligand association was possible. These results demonstrate that integrins can be functionally incorporated into peptide-tethered artificial membranes. In combination with the SPS/SPFS method, this artificial membrane system provides a reliable experimental platform for investigation of isolated membrane proteins under experimental conditions resembling those of their native environment.

Following fast adsorption processes with surface plasmon spectroscopy: reflectivity versus mismatch tracking

Surface plasmon resonance (SPR) spectroscopy is a widely used optical reflection technique for the characterization of thin films. The central quantity of SPR spectroscopy is the surface plasmon coupling angle as a characteristic signature of the prevailing interfacial architecture. Adsorption processes lead to a shift of the surface plasmon resonance which is in the thin film limit directly proportional to the corresponding mass coverage. The aim of any SPR instrument is a precise measurement of the coupling angle with a sufficient high time resolution that fast kinetic processes can be monitored. In this paper, we compare two promising methods, an established one, the reflectivity tracking and a fairly new one, the mismatch tracking. Reflectivity tracking simply records the intensity of the light in the vicinity of the coupling angle. The shift of the plasmon modifies the intensity at the detector which can subsequently be used for data analysis. Mismatch tracking is more complex. Light is focused with a lens onto the prism base and the reflected light is detected via a bicell detector. The upper and lower cell integrate over a well-defined angular range of the fan of rays produced within the focus. The mismatch in the intensity between the upper and lower segments is evaluated and used to retrieve the unknown film parameter. In this contribution we suggest some decisive variations of the originally proposed scheme and demonstrate that the modified mismatch scheme yields a significantly higher sensitivity than the original one. Furthermore, it is demonstrated that this scheme is a superior alternative to reflectivity tracking.

Electropolymerization and doping/dedoping properties of polyaniline thin films as studied by electrochemical-surface plasmon spectroscopy and by the quartz crystal microbalance

The electropolymerization and doping/dedoping properties of polyaniline ultrathin films on Au electrode surfaces were investigated by a combination of in situ electrochemical techniques, i.e., electrochemical surface plasmon spectroscopy (ESPR) and the electrochemical quartz crystal microbalance (EQCM). In the ESPR measurements, we employed two wavelengths, i.e., 632.8 and 1152 nm in order to distinguish independently the electrochromic behavior. In addition, we used spectroelectrochemical transmittance measurements in order to probe further the optical properties of the polymer films as a function of the applied potential. The real and imaginary parts of the dielectric constant of the polyaniline thin film at several doping levels was determined quantitatively by taking into consideration the thickness values obtained from the EQCM measurement. The combination of these two techniques provides a powerful method for probing the electrical, optical, and dielectric properties of conjugated ultrathin polymer films.

Investigation of non-linear optical effects at conjugated polymer/silver interface by surface plasmon resonance and surface enhanced Raman scattering

SERS experiments performed at 1064 nm with quasi-monomolecular layers of polycarbazolyldiacetylene (polyDCHD-HS: 1,6-bis-(3,6-dihexadecyl- N-carbazolyl)-2,4-hexadiyne) spun on silver-coated plates suggest that the very large value of the intensity dependent refractive index measured in such samples and at the same wavelength by surface plasmon spectroscopy (SPS) tests is related to the nanostructured surface of the silver films, that provides an electromagnetic mechanism for the enhancement of the non-linearity through local field effects at optical frequencies far from the plasmon resonance of the metal.