ABSTRACT

Surface-sensitive methods, based on surface plasmon resonance (SPR), have become increasingly interesting scientific tools in the context of biorecognition at functionalized surfaces. In the present report, we describe the development of a binding SPR assay format

based on protein-functionalized phospholipid bilayers mimicking biomembranes. As proof of principle, we incorporated type I transmembrane heterodimeric cell adhesion and signaling receptors of the integrin superfamily into phospholipid bilayers on an SPR biosensor and monitored binding of natural extracellular matrix (ECM) proteins as well as synthetic integrin ligands by SPR and its extension, surface plasmon-enhanced fluorescence spectroscopy (SPFS). This technology allows detection of binding of even very small integrin ligands, such as mono-and oligomeric Arg-Gly-Asp (RGD)-based peptides and peptidomimetics. By means of this novel binding platform, specific and sensitive recording of the association of ligands with their transmembrane receptors in a membraneous microenvironment is enabled, thereby preserving their native structural and functional properties. 20.1 Surface Plasmon Resonance and Surface

20.1.1 Principles of SPRSPR exploits optical sensors for monitoring interactions between an analyte in solution and a binding partner immobilized onto the biosensor surface. Before SPR gained popularity for the analysis of biomolecular interactions, it had already been applied for many years by material scientists for measurements of surface and optical properties of molecular films and interfaces [1, 2]. The theoretical background of SPR has been described in detail by the groups of Knoll [3] and Raether [4]. In brief, SPR occurs at the interface between two materials of different optical properties because of their different dielectric properties, for example, at the interface of a thin noble metal film in vicinity with a medium of lower dielectric properties [3, 4]. The metal layer acts as a mirror and reflects incoming light upon scanning the angle of incidence. If p-polarized incident light strikes the metal film at the resonance angle, the (nearly) free electrons within the thin conductive layer absorb energy from

the photons and start oscillating. The excited surface plasmon-polaritons, which represent surface-bound electromagnetic waves, are then propagated along the boundary between the dielectric and the metal [3]. The energy coupling during the excitation of surface plasmons is observed as a deep minimum in the reflectivity of the p-polarized light at the resonance angle of incidence [5]. The evanescent field associated with the surface plasmon wave displays its maximum in the surface and decays exponentially into the space perpendicular to it, extending into the metal and the dielectric medium [3-5]. An important property of SPR is that the intensity of the evanescent electromagnetic field is strongly enhanced when the reflectivity reaches a minimum as soon as the incident light strikes the metal surface at the resonance angle [4, 5]. When biomolecular recognition elements, which are immobilized on the surface of the metal, recognize and capture a soluble analyte, a local increase in the refractive index at the metal surface is produced. Since the position of the resonance angle is altered upon binding of macromolecules to the metal layer [5], surface plasmons, consequently, are sensitive to changes in the optical architecture near the interface (Fig. 20.1). This property of surface plasmons represents the underlying physical principle of affinity SPR biosensors, which can thus be employed as highly sensitive sensing probes for label-free and nondestructive monitoring of interfacial properties and binding processes [5, 6].