Localized surface plasmon resonance spectroscopy near molecular resonances

The peak location of the localized surface plasmon resonance (LSPR) of noble metal nanoparticles is highly dependent upon the refractive index of the nanoparticles' surrounding environment. In this study, new phenomena are revealed by exploring the influence of interacting molecular resonances and nanoparticle resonances. The LSPR peak shift and line shape induced by a resonant molecule vary with wavelength. In most instances, the oscillatory dependence of the peak shift on wavelength tracks with the wavelength dependence of the real part of the refractive index, as determined by a Kramers-Kronig transformation of the molecular resonance absorption spectrum. A quantitative assessment of this shift based on discrete dipole approximation calculations shows that the Kramers-Kronig index must be scaled in order to match experiment.     

Biological sensing using transmission surface plasmon resonance spectroscopy

Ultrathin gold island films evaporated on transparent substrates offer promising transducers for chemical and biological sensing in the transmission surface plasmon resonance (T-SPR) mode. In the present work, the applicability of T-SPR-based systems to biosensing is demonstrated, using a well-established biological model system. Au island films were evaporated on polystyrene slides and modified with a biotinylated monolayer via a multistep surface reaction, the latter assisted by the good adhesion of metal islands to polystyrene. The biotin-derivatized Au island film was then used as a biological recognition surface for selective sensing of avidin binding, distinguishing between specific and nonspecific binding to the substrate. Transduction of the binding event into an optical signal was achieved by T-SPR spectroscopy, using plasmon intensity measurements, rather than wavelength change, for maximal sensitivity and convenience. T-SPR spectroscopy of Au island films is shown to be an effective tool for monitoring the binding of biological molecules to receptor layers on the Au surface and a promising approach to label-free optical biosensing.     

Modeling of vapor sorption in polymeric film studied by surface plasmon resonance spectroscopy

Sorption process by surface plasmon resonance (SPR) was studied by exposing polymeric film made from anthracene labeled poly(methyl methacrylate) (An-PMMA) chains to various concentrations of saturated chloroform vapor. It was observed that the reflectivity changes were fast and reversible. The changes in reflectivity implied the swelling behavior of polymeric film during adsorbtion and can be explained by capturing of chloroform molecules. When clean air is introduced into gas cell similar behavior is observed but this time in the opposite direction as a result of desorption. Fick's law for diffusion was used to quantify real time SPR data for the swelling and desorption processes. It was observed that diffusion coefficients (D(s)) for swelling obeyed the t(1/2) law and found to be correlated with the amount of chloroform content in the cell. Diffusion coefficients (D(d)) during desorption were also measured and found to be increased as the saturated chloroform vapor content is increased in the cell.     

Rapid analysis of coumarins using surface plasmon resonance

Coumarin molecules are ubiquitous in nature. Several have come to prominence as potential clinical therapeutic candidates. The principal example is warfarin, which is a very widely prescribed anticoagulant. Other coumarin derivatives, such as aflatoxin B1, are insidious contaminants in crop-derived foodstuffs. Extreme potency is a common feature of all biochemically active coumarins and, thus reliable methods for their rapid and sensitive detection are of paramount importance. Accordingly, this review examines the current methods used in the analysis of these molecules and compares them with immunoassay-based strategies. As a case study, we report on our experiences with using coumarin-specific polyclonal, monoclonal, and recombinant antibodies in conjunction with a surface plasmon resonance-based biosensor for analysis of coumarins. We chart the assay development process and demonstrate high sensitivity and reproducibility that compares favorably with established methodologies.     

Mechanism of G-protein activation by rhodopsin

Rhodopsin is a member of the family of G-protein-coupled receptors (GPCRs), and is an excellent molecular switch for converting light signals into electrical response of the rod photoreceptor cells. Light initiates cis-trans isomerization of the retinal chromophore of rhodopsin and leads to the formation of several thermolabile intermediates during the bleaching process. Recent investigations have identified spectrally distinguishable two intermediate states that can interact with the retinal G-protein, transducin, and have elucidated the functional sharing of these intermediates. The initial contact with GDP-bound G-protein occurs in the meta-Ib intermediate state, which has a protonated Schiff base as its chromophore. The meta-Ib intermediate in the complex with the G-protein converts to the meta-II intermediate with releasing GDP from the alpha-subunit of the G protein. Meta-II has a de-protonated Schiff base chromophore and induces binding of GTP to the alpha-subunit of the G-protein. Thus, the GDP-GTP exchange reaction, namely G-protein activation, by rhodopsin proceeds through at least two steps, with conformational changes in both rhodopsin and the G-protein.     

Characterization of pore formation by streptolysin O on supported lipid membranes by impedance spectroscopy and surface plasmon resonance spectroscopy

We report the study of the interactions of bacterial toxin streptolysin O (SLO) and cholesterol-containing membranes using electrochemical impedance and surface plasmon resonance (SPR) spectroscopy at low hemolytic units on a novel supported membrane interface. The detailed understanding of the process will aid significantly the construction of nanoscale transport channels for biosensing applications. Cholesterol-containing egg PC vesicles, pristine and incubated with SLO toxin, were fused onto a hexyl thioctate (HT)-modified gold substrate. The charge-transfer resistance of the resulting lipid membrane, which is related to the formation of the transmembrane pores, is measured with the aid of an electroactive probe. Impedance spectra were collected over a range of 0.1-100 kHz, and the obtained complex resistance was fit to an equivalent circuit. The charge-transfer resistance decreases for increasing SLO concentration, following a first-order exponential decay. The two-part membrane interface was further characterized with SPR spectroscopy. For the hexyl thioctate support layer, an equivalent monolayer thickness of 1.3 nm was determined. This value suggests a loosely packed structure of the monolayer on gold, presenting an ideal platform for permeability studies. A comparative study on the fusion behavior of vesicles with and without SLO induced pores revealed no substantial difference for the two systems, indicating that the pore formation has no adverse effect on the integrity of the vesicles. The resulting lipid membrane thickness from pre-perforated lipids was found to be 3.2 nm, suggesting that one leaflet is knocked off during the fusion process and a hybrid membrane is formed. A slightly higher thickness value of 3.4 nm was obtained for membranes from non-perforated vesicles. Deposition of lipids and subsequent incubation with SLO, as monitored by SPR, shows that the HT surface chemistry allows partial insertion of the toxin into the membrane, indicating unique properties as compared to the previously explored long-chain alkylthiols.     

Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis.

A system for real-time biospecific interaction analysis using biosensor technology based on the optical phenomenon surface plasmon resonance is described. The biospecific interface is a sensor chip covered with a hydrogel matrix. One component of the interaction to be studied is immobilized covalently to the hydrogel and other interactants are passed over the chip in solution. The mass change at the sensor surface, reflecting the progress of the interaction studied, is monitored in real time. The technique, which does not require molecular labels for detection, can measure mass changes down to 10 pg/mm2. Repeated analyses can be performed on the same sensor chip. Applications shown include kinetic measurements, binding site analysis and concentration determination.     

Recent progress in the studies of electrochemical interfaces by surface plasmon resonance spectroscopy and microscopy

Surface plasmon resonance (SPR) is a label-free spectroscopic technique that is highly sensitive to various surface reactions. Incorporating SPR into electrochemical measurements has emerged as a powerful method to study both faradaic and non-faradaic processes. SPR microscopy (SPRM) integrates an optical microscope into SPR detection, which further offers high throughput detection and spatially resolved information at an electrode surface and thus, has attracted attention especially in single entity electrochemical studies. In this review, the progress in the studies of electrochemical interfaces by SPR and SPRM during the past two years will be discussed.     

Ultrathin molecularly imprinted polymer sensors employing enhanced transmission surface plasmon resonance spectroscopy

An ultrathin novel nanosensor (31.5 +/- 4.1 nm thick in the absence of analytes), employing a molecularly imprinted polymer as a recognition element for cholesterol and gold nanoparticle enhanced transmission surface plasmon resonance spectroscopy for detection, was constructed.     

Determination of the surface pK of carboxylic- and amine-terminated alkanethiols using surface plasmon resonance spectroscopy

When using self-assembled monolayers (SAMs) with ionizable functional groups, such as COOH and NH2, the dissociation constant (pKd) of the surface is an important property to know, since it defines the charge density of the surface for a given bulk solution pH. In this study, we developed a method using surface plasmon resonance (SPR) spectroscopy for the direct measurement of the pKd of a SAM surface by combining the ability of SPR to detect the change in mass concentration close to a surface and the shift in ion concentration over the surface as a function of surface charge density. This method was then applied to measure the pKd values of both COOH- and NH2-functionalized SAM surfaces using solutions of CsCl and NaBr salts, respectively, which provided pKd values of 7.4 and 6.5, respectively, based on the bulk solution pH. An analytical study was also performed to theoretically predict the shape of the SPR plots by calculating the excess mass of salt ions over a surface as a function of the difference between the solution pH and surface pKd. The analytical relationships show that the state of surface charge also influences the local hydrogen ion concentration, thus resulting in a substantial local shift in pH at the surface compared to the bulk solution as a function of the difference between the bulk solution pH and the pKd of the surface.     

In-situ analysis of stepwise self-assembled 1,6-hexanedithiol multilayers by surface plasmon resonance measurements

1,6-Hexandithiol (HDT) forms 6.9 +/- 1.0 A thick defect-free monolayers on gold substrates if the solution is purged by argon during the adsorption while long term (> 1000 min) exposure of the substrate to alcoholic HDT results in the stepwise formation of multilayers in the absence of argon purging.     

Layer-by-layer self-assembly preparation of layered double hydroxide/polyelectrolyte nanofilms monitored by surface plasmon resonance spectroscopy

Layer-by-layer self-assembly was used to prepare nanofilms of (2:1) MgAl-layered double hydroxide (LDH) nanoparticles and polyacrylic acid or sodium polystyrene sulfonate. The multilayers were attached to ~50-nm thick gold films on microscopy glass slides prepared by vacuum evaporation. The contact between the gold film and the multilayered films was mediated via surface modification with thiols, adsorption of poly(diallyl dimethyl ammonium) chloride (PDDA) or direct binding of the LDH particles. Surface plasmon resonance (SPR) spectra of the multilayered films were analyzed by fitting the Fresnel equations. The shifts in the SPR angle (_SPR) due to the adsorption/deposition on the gold surface were used to evaluate the process of building up the multilayers. Strong surface/multilayer contact formed when electrostatic attraction and hydrophobic interaction were combined as in the case of mercaptopropanoic acid or PDDA sticking layers. The LDH suspension concentration strongly influenced the number of deposited layers. The multilayer films were investigated by reflection FT-IR spectroscopy.     

Characterization of surface-confined alpha-synuclein by surface plasmon resonance measurements

Urea-driven denaturation and renaturation of surface-bound alpha-synuclein are monitored by surface plasmon resonance (SPR) spectroscopy. The differential SPR angle shift (Delta Theta(SPR))(Net) enables us to estimate the Gibbs free energy change (DeltaG(o)) for the denaturation of the supported alpha-synuclein. DeltaG(o) for the denaturation of the supported alpha-synuclein, which is indirectly related to its biological activity can be increased significantly by the mixed self-assembled monolayers of 11-mercaptoundecanoic acid and 1,6-hexanedithiol. These SPR measurements of surface-bound biomolecules suggested herein can be further utilized to design effective biological scaffold for biosensor, biocatalyst, and possible diagnosis.     

In situ sensing of metal ion adsorption to a thiolated surface using surface plasmon resonance spectroscopy

The kinetics of the adsorption of metal ions onto a thiolated surface and the selective and quantitative sensing of metal ions were explored using surface plasmon resonance (SPR) spectroscopy. The target metal ion was an aqueous solution of Pt2+ and a thin-gold-film-coated glass substrate was modified with 1,6-hexanedithiol (HDT) as a selective sensing layer. SPR spectroscopy was used to examine the kinetics of metal ion adsorption by means of the change in SPR angle. The selectivity of the thiolated surface for Pt2+ over other divalent metal ions such as Cu2+, Ni2+, and Cd2+ was evident by the time-resolved SPR measurement. SPR angle shift, deltatheta(SPR), was found to increase logarithmically with increasing concentration of Pt2+ in the range of 1.0 x 10(-5)-1.0 mM. The rate of Pt2+ adsorption on HDT observed at both 0.1 and 1 mM Pt2+ accelerates until the surface coverage reaches approximately 17%, after which the adsorption profile follows Langmuirian behavior with the surface coverage. The experimental data indicated that heavy metal ions were adsorbed to the hydrophobic thiolated surface by a cooperative mechanism. A mixed self-assembled monolayer (SAM) composed of HDT and 11-mercaptoundecanoic acid was used to reduce the hydrophobicity of the thiol-functionalized surface. The addition of hydrophilic groups to the surface enhanced the rate of adsorption of Pt2+ onto the surface. The findings show that the adsorption of metal ions is strongly dependent upon the hydrophilicity/hydrophobicity of the surface and that the technique represents an easy method for analyzing the adsorption of metal ions to a functionalized surface by combining SPR spectroscopy with a SAM modification.     

Detection of transglucosidase-catalyzed polysaccharide synthesis on a surface in real time using surface plasmon resonance spectroscopy

Biological systems that involve enzyme catalysis at surfaces, particularly strategically important ones that involve insoluble substrates/products such as the cell wall and the starch granule, require analyses beyond classical solution state enzymology. Using a model system, we have demonstrated the real-time measurement of transglucosidase activity on a surface using surface plasmon resonance (SPR) spectroscopy. We monitored the extension of a (partially carboxymethylated) dextran surface with alternansucrase and sucrose as a glycosyl donor. Conditions were used where surface polymer synthesis rates were a function of enzyme concentration and proportional to the extent of enzyme binding to the surface. A method to determine the turnover number of the enzyme on the surface was also developed. The presence of a new amorphous polysaccharide was observed optically, detected by lectin binding and imaged by atomic force microscopy. This surface method will have utility in a wide range of carbohydrate enzyme systems including screens.     

Characterization of oxidoreductase-redox polymer electrostatic film assembly on gold by surface plasmon resonance spectroscopy and Fourier transform infrared-external reflection spectroscopy

The electrostatic assembly of nanocomposite thin films consisting of alternating layers of an organometallic redox polymer (RP) and oxidoreductase enzymes, glucose oxidase (GOX), lactate oxidase (LOX) and pyruvate oxidase (PYX), was investigated. Multilayer nanostructures were fabricated on gold surfaces by the deposition of an anionic self-assembled monolayer of 11-mercaptoundecanoic acid, followed by the electrostatic attachment of a cationic RP, poly(vinylpyridine Os(bis-bipyridine)₂Cl-co-allylamine) (PVP-Os-AA), and anionic oxidoreductase enzymes. Surface plasmon resonance (SPR) spectroscopy, Fourier transform infrared external reflection spectroscopy (FT-IR-ERS) and electrochemistry were employed to characterize the assembly of these nanocomposite films. The surface concentration of GOX was found to be 2.4 ng/mm² for the first enzyme layer and 1.96 ng/mm² for the second enzyme layer, while values of 10.7 and 1.3 ng/mm² were obtained for PYX and LOX, respectively. The apparent affinity constant for GOX adsorption was found to be 8×10⁷ M⁻¹. FT-IR-ERS was used to verify the incorporation of GOX and its conformational stability inside of these nanocomposite thin films. An SPR instrument with a flow-through cell was modified by additions of Ag/AgCl reference and Pt counter electrodes, with the gold-coated SPR surface film serving as the working electrode. This enabled real-time observation of the assembly of sensing components and immediate, in situ electrochemical verification of substrate-dependent current upon the addition of enzyme to the multilayer structure. A glucose-dependant amperometric response with sensitivity of 0.197 μA/cm²/mM for a linear range of 1-10 mM of glucose was obtained. The SPR and FT-IR-ERS studies also showed no desorption of polymer or enzyme from the nanocomposite RP-GOX structure when stored in aqueous environment occurred over the period of 3 weeks, suggesting that decreasing substrate sensitivity with time was due to loss of enzymatic activity rather than loss of film compounds from the nanostructure.     

Wide-field single metal nanoparticle spectroscopy for high throughput localized surface plasmon resonance sensing

Noble metal nanoparticles (mNPs) have a distinct extinction spectrum arising from their ability to support Localized Surface Plasmon Resonance (LSPR). Single-particle biosensing with LSPR is label free and offers a number of advantages, including single molecular sensitivity, multiplex detection, and in vivo quantification of chemical species etc. In this article, we introduce Single-particle LSPR Imaging (SLI), a wide-field spectral imaging method for high throughput LSPR biosensing. The SLI utilizes a transmission grating to generate the diffraction spectra from multiple mNPs, which are captured using a Charge Coupled Device (CCD). With the SLI, we are able to simultaneously image and track the spectral changes of up to 50 mNPs in a single (～1 s) exposure and yet still retain a reasonable spectral resolution for biosensing. Using the SLI, we could observe spectral shift under different local refractive index environments and demonstrate biosensing using biotin-streptavidin as a model system. To the best of our knowledge, this is the first time a transmission grating based spectral imaging approach has been used for mNPs LSPR sensing. The higher throughput LSPR sensing, offered by SLI, opens up a new possibility of performing label-free, single-molecule experiments in a high-throughput manner.