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Content archived on 2024-05-29

Improving the detection sensitivity for protein micro-arrays: wave-guide SPR imaging and nano-particle enhanced imaging

Final Activity Report Summary - IMPSENS (Improving the detection sensitivity for protein microarrays: waveguide SPR imaging and nanoparticle enhanced imaging)

Surface plasmon resonance microscopy (SPRM) is an emerging technique for the observation of surface phenomena on the molecular scale with unrivalled sensitivity. A surface plasmon is a collective oscillation of surface bound electrons in a nobel metal, such as gold. Surface plasmons are excited by photons, under conservation of momentum and energy. Technically, p-polarised light is passed through a coupling arrangement onto a semitransparent thin gold layer and the reflected light, i.e. the light that is not coupled into surface plasmons is analysed with a digital camera. The coupling conditions, such as angle of incidence and wavelength of the light can be used to describe the medium adherent to the metal film. In contrast to fluorescence microscopy SPRM does not require labelling with flurophores, the observed contrast is based on the refractive index only. This is a tremendous advantage, since it allows the observations of artificial lipid membranes, proteins and DNA in the native, unaltered state.

A particular application is the observation of protein-protein interactions to determine, for example the affinity between antibodies and antigens. One partner in the complex is immobilised on the metal surface, by covalent linkage and the ligand is flushed across the surface. Upon binding between the partners, the total surface bound mass is increased and this signal can easily be measured in real time. This allows it to describe the interaction strength and dynamic between a virtually limitless numbers of biomolecules and provides a platform for studying complex biological systems in the laboratory. Utilising wide field microscopy it is possible to deposit hundredths of different proteins or DNA samples in microspots and test complex mixtures of analytes. This allows high throughput screening for the potential diagnosis of diseases and genetic defects.