Surface Plasmon Resonance Curve Research Articles

Determination of receptor-ligand kinetic and equilibrium binding constants using surface plasmon resonance: application to the lck SH2 domain and phosphotyrosyl peptides.

Experimental and computational methods were developed for surface plasmon resonance (SPR) measurements involving interactions between a solution-binding component and a surface-immobilized ligand. These protocols were used to distinguish differences in affinity between the SH2 domain of lck and phosphotyrosyl peptides. The surface-immobilized ligand was the phosphotyrosyl peptide EPQpYEEIPIA, which contains a consensus sequence (pYEEI) for binding lck SH2. In the kinetic experiment, SPR phenomena were measured during association and dissociation reactions for a series of glutathione-S-transferase (GST)-SH2 concentrations, generating a set of SPR curves. A global computational analysis using an A + B<==>AB model resulted in single set of parameter estimates and statistics. In an abbreviated format, an equilibrium experiment was designed so that equilibrium constants (Keq) could be determined rapidly and accurately. A competitive equilibrium assay was developed for GST-SH2 in which Keq values for a series of phosphotyrosyl peptides (derived from the pYEEI sequence) varied over 3 orders of magnitude. Interestingly, these results highlighted the significance of the +1 glutamate in providing high-affinity binding to the SH2 domain. For most drug discovery programs, these Keq determinations are a sufficient measure of potency for the primary screen, with koff and kon determined in a secondary assay. Thus, the application of these techniques to SPR binding phenomena should prove valuable in the discovery and design of receptor-ligand antagonists.

Assembly and molecular organization of self-assembled lipid bilayers on solid substrates monitored by surface plasmon resonance spectroscopy

The structural properties of lipid films, made from a squalene/butanol solution containing varying amounts (0–15 mg/ml) of egg phosphatidylcholine and deposited on a thin metallic silver layer, were investigated using surface plasmon resonance (SPR) spectroscopy. Optical parameters (thickness, refractive index and extinction coefficient) of such supported self-assembled lipid membranes were obtained from a theoretical analysis of the experimental SPR curves. The mass of the lipid membrane and the area and volume occupied by one lipid molecule were also calculated. The results were consistent with the formation of durable and homogeneous lipid bilayers on the solid substrate, and indicated similarities in structural properties between the present lipid bilayers and freely suspended and Langmuir-Blodgett bilayer membranes. Such bilayers represent a simple model for biological membranes, as well as providing a means of immobilizing proteins for various practical applications, including receptor-based sensors and molecular devices. The results confirm the value of the SPR technique for investigating the properties of thin biomolecular dielectric films deposited on a metal surface.

Biosensors based on surface plasmons excited in non-noble metals

A novel idea of utilizing transition metals in surface plasmon resonance detection has been verified both theoretically and experimentally. Different metal films were covered with increasing numbers of monomolecular layers of cadmium behenate and surface plasmon resonance curves were recorded for each additional layer. Results of the measurements show that it is the position of the ‘down-way’ slope of the resonance curve that is most affected by changes in the resonance conditions, not the width of the whole curve nor the position of the minimum. The angle of the slope shifts to higher values when the number of monomolecular layers increases. Thus, the sensitivity of the system depends on the first derivative of the resonance curve (‘steepness’ of the slope). For example, detection sensitivities obtained with such transition metals as palladium are comparable with those obtained with gold. These results were confirmed by computer simulations of the surface plasmon resonance phenomenon. For calculations the Fresnel reflections equations for multilayered media were used, with exact solutions obtained numerically.