Inhibition binding studies of glycodendrimer/lectin interactions using surface plasmon resonance

Understanding protein/carbohydrate interactions is essential for elucidating biological pathways and cellular mechanisms but is often difficult due to the prevalence of multivalent interactions. Here, we evaluate the multivalent glycodendrimer framework as a means to describe the inhibition potency of multivalent mannose-functionalized dendrimers using surface plasmon resonance (SPR). Using highly robust, mannose-functionalized dithiol self-assembled monolayers on gold surfaces, we found that glycodendrimers were efficient inhibitors of protein/carbohydrate interactions. IC₅₀ values ranging from 260 nM to 13 nM were obtained for mannose-functionalized dendrimers with Concanavalin A.     

Antigen-antibody interactions: binding studies with fluorescence and surface plasmon resonance exemplified by acid-traseolide as antigen

The interaction between the musk fragrance acid-traseolide and monoclonal antibodies (mAB) generated against this odorant has been investigated with two different techniques. Fluorescence spectroscopy was used to study the quenching of tryptophan fluorescence of the antibody upon binding acid-traseolide. This spectroscopic approach is based on measurements under equilibrium conditions. The second technique exploited the surface plasmon resonance (SPR) phenomenon. The acid-traseolide was immobilized in the surface matrix and upon presenting mAB changes in SPR were recorded in real time during the association reaction. The SPR approach can be considered as a kinetic method. Although having a different origin, both methods lead to comparable equilibrium dissociation constants (Kd). However, the results obtained with fluorescence spectroscopy were more accurate and reproducible. Not only the association of acid-traseolide with antibody was evaluated, also Fab fragment and peptide (H3-peptide) mimicking the heavy chain CDR3 of this antibody were included in this study. The Kd-values, determined by both methods, increase in the order mAB < Fab < H3-peptide because of diminishing recognition.     

Carbohydrate-protein interactions at interfaces: comparison of the binding of Ricinus communis lectin to two series of synthetic glycolipids using surface plasmon resonance studies

Two C-lactosyl lipids and the related C-galactosyl lipids have been synthesised and their binding to RCA120 plant lectin was compared with a second series of thiolactosylethoxyalkanes. The interactions were measured quantitatively in real time by surface plasmon resonance (BIAcore) at a range of concentrations and temperatures from 5 to 30 degrees C. The C-galactosyl lipid (1,3-dimethyl-5-[beta-D-galactopyranosyl]-5-(4-octadecyloxybenzyl)pyrimidine-2,4,6-trione) bound much more weakly with a K(A) = 8.86 x 10(5) than the corresponding C-lactosyl lipid (1,3-dimethyl-5-[beta-D-galactopyranosyl-(1 --> 4)-beta-D-glucopyranosyl]-5-(4-octadecyloxybenzyl)pyrimidine-2,4,6-trione) (K(A) = 2.31 x 10(7)). The influence of the linker region of the two different series of lactosyl lipids was clearly demonstrated by the differences in the binding to RCA120 lectin. The changes in kinetic values and in the enthalpic and entropic contribution to the free energy of binding reflected the importance of the linker and the hydrocarbon anchor holding the synthetic glycolipids in the neomembrane.     

Screening nucleotide binding to amino acid-coated supports by surface plasmon resonance and nuclear magnetic resonance

Here, we describe a rapid and efficient screening method using surface plasmon resonance (SPR) and saturation transfer difference–nuclear magnetic resonance (STD-NMR) spectroscopy to yield information regarding the residues involved in nucleotide binding to amino acid-coated supports. The aim of this work was to explore the use of these spectroscopic techniques to study amino acid–nucleotide interactions in order to improve the binding specificity of the amino acid ligands used to purify plasmid DNA. For SPR, we present a strategy that immobilizes arginine and lysine on a surface as model supports, and we analyze binding responses when synthetic homo-deoxyoligonucleotides are injected over the amino acid surface. The binding responses are detectable and reproducible despite the small size of the immobilized amino acids. Using STD-NMR, we performed epitope mapping of homo-deoxyoligonucleotides bound to l-arginine–bisoxyran–Sepharose and l-lysine–Sepharose supports. Polynucleotide binding preferences differed; for example, polyC interacted preferentially through its backbone with the two supports, whereas polyT bound the supports through its thymine moiety. STD-NMR combined with SPR measurements was successfully used to screen amino acid–nucleotide interactions and determine the binding affinities of the complexes.     

Characterization of collagen thin films for von Willebrand factor binding and platelet adhesion

Von Willebrand factor (VWF) binding and platelet adhesion to subendothelial collagens are initial events in thrombus formation at sites of vascular injury. These events are often studied in vitro using flow assays designed to mimic vascular hemodynamics. Flow assays commonly employ collagen-functionalized substrates, but a lack of standardized methods of surface ligation limits their widespread use as a clinical diagnostic. Here, we report the use of collagen thin films (CTF) in flow assays. Thin films were grown on hydrophobic substrates from type I collagen solutions of increasing concentration (10, 100, and 1000 μg/mL). We found that the corresponding increase in fiber surface area determined the amount of VWF binding and platelet adhesion. The association rate constant (k(a)) of plasma VWF binding at a wall shear stress of 45 dyn/cm(2) was 0.3 × 10(5), 1.8 × 10(5), and 1.6 × 10(5) M(-1) s(-1) for CTF grown from 10, 100, and 1000 μg/mL solutions, respectively. We observed a 5-fold increase in VWF binding capacity with each 10-fold increase in collagen solution concentration. The association rates of Ser1731Thr and His1786Asp VWF mutants with collagen binding deficiencies were 9% and 22%, respectively, of wild-type rates. Using microfluidic devices for blood flow assays, we observed that CTF supported platelet adhesion at a wall shear rate of 1000 s(-1). CTF grown from 10 and 100 μg/mL solutions had variable levels of platelet surface coverage between multiple normal donors. However, CTF substrates grown from 1000 μg/mL solutions had reproducible surface coverage levels (74 ± 17%) between normal donors, and there was significantly diminished surface coverage from two type 1 von Willebrand disease patients (8.0% and 24%). These results demonstrate that collagen thin films are homogeneous and reproducible substrates that can measure dysfunctions in VWF binding and platelet adhesion under flow in a clinical microfluidic assay format.     

Kinetic studies on the interactions between glycolipid biosurfactant assembled monolayers and various classes of immunoglobulins using surface plasmon resonance

Kinetic studies on the interactions between self-assembled monolayers of mannosylerythritol lipids (MELs), which are glycolipid biosurfactants abundantly produced by microorganisms, and various classes of immunoglobulins including human IgG, IgA, and IgM were performed using surface plasmon resonance (SPR). The effect of the MEL structure on the binding behavior of HIgG was examined. Assembled monolayers of MEL-A having two acetyl groups on the headgroup gave a high affinity (K_d = 1.7 × 10⁻⁶ M) toward HIgG, while those of MEL-B or MEL-C having only one acetyl group at C-6′ or C-4′ position gave little affinity. Our kinetic analysis revealed that the binding manner of HIgG, HIgA (K_d = 2.4 × 10⁻⁷ M), and HIgM (K_d = 2.2 × 10⁻⁷ M) to the assembled monolayers of MEL-A is not the monovalent mode but the bivalent mode, and both the first and second rate association constants (k_a1, k_a2) increase with an increase in the number of antibody binding sites, while those for dissociation (k_d1, k_d2) changed little. Moreover, we succeeded in directly observing great amounts of HIgG, HIgA, and HIgM bound to MEL-A monolayers using atomic force microscopy (AFM). Finally, we found that MEL-A assembled monolayer binds toward various IgG derived from mouse, pig, rabbit, horse, goat, rat, and bovine as well as human IgG (HIgG), and the only exception was sheep IgG. These results clearly demonstrate that MEL-A assembled monolayers would be useful as noble affinity ligand system for various immunoglobulins.     

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.     

Electrochemical surface plasmon resonance investigation of dodecyl sulfate adsorption to electroactive self-assembled monolayers via ion-pairing interactions

The redox-induced assembly of amphiphilic molecules and macromolecules at electrode surfaces is a potentially attractive means of electrochemically modulating the organization of materials and nanostructures on solid substrates via ion-pairing interactions or charge-transfer complexation. In this regard, we have investigated the potential-induced adsorption and aggregation of dodecyl sulfate, a common anionic surfactant, at a ferrocenylundecanethiolate (FcC11SAu) self-assembled monolayer (SAM)/aqueous solution interface by electrochemical surface plasmon resonance (ESPR) spectroscopy. The surfactant anions adsorb onto the electroactive SAM by specific ion-pairing interactions with the oxidized ferricinium species. The ferricinium charge density (QFc+) obtained by cyclic voltammetry and surface coverage measured by SPR indicate that the dodecyl sulfate forms an interdigitated monolayer, where half of the surfactant molecules have their sulfate headgroups paired to the surface and half have their headgroups exposed to the aqueous solution. The surface coverage of dodecyl sulfate was found to depend on both the ferricinium surface concentration and the surfactant aggregation state in solution. A maximum coverage of dodecyl sulfate on the ferricinium surface is obtained below the critical micelle concentration (cmc), in contrast to dodecyl sulfate adsorption to SAM surfaces of static positive charge. This marked difference in adsorption behavior is attributed to the dynamic generation of ferricinium by potential cycling and the specific nature of the ion-pairing interactions versus pure electrostatic ones. The results presented point to a new way of organizing molecules via electrical stimulus.     

Application of surface plasmon resonance spectroscopy for adsorption studies of different types of components on poly(dimethylsiloxane)

Surface plasmon resonance (SPR) spectroscopy is utilized to study in real-time and, by label-free means, the reversible and quasi-irreversible adsorption of small ionic or neutral molecules, pharmaceuticals, and proteins on poly(dimethylsiloxane) (PDMS) surfaces. The SPR sensor is covered with 0.2% (w/v) PDMS in octane. During the timescale of a typical lab-on-a-chip analysis or an electrophoretic separation, it was found that small neutral components containing a hydrophobic part do not adsorb or absorb onto PDMS, while larger, water-soluble polymer-like materials like proteins generally irreversibly adsorb to PDMS. The technique can be used to monitor the kinetics of adsorption and desorption of the molecules. For the non-specific adsorption of teicoplanin to PDMS, a Langmuir-like adsorption isotherm was obtained (K_d = 32 ± 2 μmol L⁻¹).     

Use of surface plasmon resonance to study the adsorption of detergents on poly(dimethylsiloxane) surfaces

This paper demonstrates the use of surface plasmon resonance to study adsorption (either reversible or irreversible) of detergents on PDMS surfaces in real time. The surface plasmon resonance measurements can directly provide information about the adsorption/desorption processes of detergents on the surface revealing the durability of the adsorbed layer and the anticipated degree of the EOF. Hydroxypropyl methylcellulose very strongly adsorbs onto PDMS and can be considered both a semipermanent layer and stable semipermanent coating. Adsorbed SDS or CTAB layers were stable for several minutes upon rinsing the surface with solution not containing the detergent. It was shown that SDS coated onto PDMS in microchips has the potential to afford similar separations in PDMS as found in conventional fused silica capillaries.     

Kinetic discrimination of sequence-specific DNA-drug binding measured by surface plasmon resonance imaging and comparison to solution-phase measurements

We demonstrate the use of surface plasmon resonance (SPR) imaging for direct detection of small-molecule binding to surface-bound DNA probes. Using a carefully designed array surface, we quantitatively discriminate between the interactions of a model drug with different immobilized DNA binding sites. Specifically, we measure the association and dissociation intercalation rates of actinomycin-D (ACTD) to and from double-stranded 5'-TGCT-3' and 5'-GGCA-3' binding sites. The rates measured provide mechanistic information about the DNA-ACTD interaction; ACTD initially binds nonspecifically to DNA but exerts its activity by dissociating slowly from strong affinity sites. We observe a slow dissociation time of kd-1 = 3300 +/- 100 s for ACTD bound to the strong affinity site 5'-TGCT-3' and a much faster dissociation time (210 +/- 15 s) for ACTD bound weakly to the site 5'-GGCA-3'. These dissociation rates, which differ by an order of magnitude, determine the binding affinity for each site (8.8 x 10(6) and 1.0 x 10(6) M(-1), respectively). We assess the effect the surface environment has on these biosensor measurements by determining kinetic and thermodynamic constants for the same DNA-ACTD interactions in solution. The surface suppresses binding affinities approximately 4-fold for both binding sites. This suppression suggests a barrier to DNA-drug association; ACTD binding to duplex DNA is approximately 100 times slower on the surface than in solution.     

Multichannel surface plasmon resonance imaging and analysis of micropatterned self-assembled monolayers and protein affinity interactions

Multichannel images of 11-mercaptoundecanoic acid and 11-mercapto-1-undecanol self-assembled monolayers together with a biospecific interferon-gamma (IFN-gamma)/anti-IFN-gamma antibody immunoaffinity interaction were observed by the two-dimensional surface plasmon resonance (2D-SPR) imaging system. With the fabricated 2D-SPR imaging system, adopting a white light source in combination with a narrow band-pass filter, sharp images were resolved, minimizing the diffraction patterns on the resulting images. Micropatterning of self-assembled monolayers was acheived by exploiting the UV photolysis of thiol bonding, instead of conventional photolithography. The line profile calibration of the image contrast with ellipsometric analysis enabled us to discriminate the change in monolayer thickness within a sub-nanometer scale. For the protein interactions on the surface, the biospecific affinity recognition reaction of IFN-gamma antigen with surface-immobilized antibody was analyzed. Through the signal amplification strategy based on the enzyme-catalyzed precipitation reaction in a sandwich-type immunoassay, biospecific antigen binding was found detectable down to a concentration of 1 ng/mL.     

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.     

Fabrication of novel molecular recognition membranes by physical adsorption and self-assembly for surface plasmon resonance detection of TNT

Recent concern on international terrorism and weapons of mass destruction demands the development of novel analytical methods for identification and quantification of explosive molecules. In this article, we describe the development of high-performance immunosensors for detection of 2,4,6-trinitrotoluene (TNT), a prime component of the landmines and bombs used by terrorist and military forces. The immunosensors were constructed by physical adsorption and self-assembly methods, and their binding interactions with a monoclonal anti-TNT antibody were evaluated for TNT detection using the surface plasmon resonance technique. A home-made 2,4,6-trinitrophenyl-keyhole limpet hemocyanine conjugate was used for physical adsorption. A poly(ethylene glycol) hydrazine hydrochloride thiolate was used in the construction of self-assembled monolayer surface and was immobilized with trinitrophenyl-β-alanine by the amide coupling method. The immunosensors were highly selective, regenerable, rapid, and exhibited remarkable sensitivity down to the parts-per-trillion level for TNT by the indirect competitive inhibition principle.     

Sensitivity of metal nanoparticle surface plasmon resonance to the dielectric environment

Electrodynamic simulations of gold nanoparticle spectra were used to investigate the sensitivity of localized surface plasmon band position to the refractive index, n, of the medium for nanoparticles of various shapes and nanoshells of various structures. Among single-component nanoparticles less than 130 nm in size, sensitivities of dipole resonance positions to bulk refractive index are found to depend only upon the wavelength of the resonance and the dielectric properties of the metal and the medium. Among particle plasmons that peak in the frequency range where the real part of the metal dielectric function varies linearly with wavelength and the imaginary part is small and slowly varying, the sensitivity of the peak wavelength, lambda, to refractive index, n, is found to be a linearly increasing function of lambda, regardless of the structural features of the particle that determine lambda. Quasistatic theory is used to derive an analytical expression for the refractive index sensitivity of small particle plasmon peaks. Through this analysis, the dependence of sensitivity on band position is found to be determined by the wavelength dependence of the real part, epsilon', of the particle dielectric function, and the sensitivity results are found to extend to all particles with resonance conditions of the form, epsilon' = -2chin(2), where chi is a function of geometric parameters and other constants. The sensitivity results observed using accurate computational methods for dipolar plasmon bands of gold nanodisks, nanorods, and hollow nanoshells extend, therefore, to particles of other shapes (such as hexagonal and chopped tetrahedral), composed of other metals, and to higher-order modes. The bulk refractive index sensitivity yielded by the theory serves as an upper bound to sensitivities of nanoparticles on dielectric substrates and sensitivities of nanoparticles to local refractive index changes, such as those associated with biomolecule sensing.     

Application of octadecanethiol self-assembled monolayer to cholesterol biosensor based on surface plasmon resonance technique

Octadecanethiol (ODT) self-assembled monolayer (SAM) prepared onto gold-coated glass plate has been modified by using nitrene reaction of 1-fluoro-2-nitro-4-azidobenzene (FNAB) that further covalently binds to cholesterol oxidase (ChOx) via thermal reaction. FNAB acts as a bridge (cross-linker) between SAM and ChOx. The ChOx/FNAB/ODT/Au electrode thus fabricated has been characterized using contact angle (CA) measurements, UV-vis spectroscopy, electrochemical techniques and X-ray photoelectron spectroscopy (XPS) technique, respectively. This ChOx/FNAB/ODT/Au bioelectrode has been utilized for estimation of cholesterol in solution using surface plasmon resonance (SPR) technique. This SPR based cholesterol biosensor has linearity from 50 to 500 mg/dl of cholesterol in solution with lower detection limit of 50 mg/dl and shelf life of about 2 months when stored at 4 °C.     

Use of high-resolution techniques for the characterization of clotting factor VIII.

Dealing with the structural characterization of clotting factor VIII (FVIII) requires the application of several high-resolution analytical techniques. Besides the analytical point of view, a detailed knowledge of FVIII structure, production and therapeutic application is necessary. This review gives an overview of most of the currently applied analytical methods and how they deal with the complex analytical problem, investigating FVIII in a sample matrix containing large amounts of accompanying plasma proteins.     

Stability of the gold/silica thin film interface: electrochemical and surface plasmon resonance studies

This article reports chemical stability studies of a gold film electrode coated with thin silicon oxide (SiOx) layers using electrochemical, surface plasmon resonance (SPR) and atomic force microscopy (AFM) techniques. Silica films with different thicknesses (d = 6.4, 9.7, 14.5, and 18.5 nm) were deposited using a plasma-enhanced chemical vapor deposition technique (PECVD). For SiOx films with d >/= 18.5 nm, the electrochemical behavior is characteristic of a highly efficient barrier for a redox probe. SiOx films with thicknesses between 9.5 and 14.5 nm were found to be less efficient barriers for electron transfer. The Au/SiOx interface with 6.4 nm of SiOx, however, showed an enhanced steady-state current compared to that of the other films. The stability of this interface in solutions of different pH was investigated. Whereas a strongly basic solution led to a continuous dissolution of the SiOx interface, acidic treatment produced a more reticulated SiOx film and improved electrochemical behavior. The electrochemical results were corroborated by SPR measurements in real time and AFM studies.