Surface plasmon resonance (SPR) techniques have been widely used for determining the affinity between proteins and also as a biosensor platform for measuring analyte concentrations based on bioreceptor-ligand interactions. One of the primary features of SPR methods that make them so useful is that they do not require special reagents (tags or labels) for detecting the extent of binding – they are reagentless methods. However one of the shortcomings of the many commercial devices that utilize the SPR effect is that they use some modification of a total internal reflection mode (Kretschmann configuration). In this mode it is difficult to develop array-type sensors where hundreds of different bioreceptors are placed on a surface in a defined pattern allowing for a variety of high-throughput screening methods. The method described here is based on a phenomenon known as localized surface plasmon resonance. This behavior is exhibited by nano-sized holes in thin gold films. A typical preparation consists of random nanoholes in a transparent gold layer. This perforated coating is achieved by first depositing negatively charged polystyrene nanoparticles onto positively charged microscope glass slides functionalized with amine groups. Then, a ~20 nm thick Au film is thermally evaporated onto the glass slides. The particles are then stripped from the slide using an adhesive tape, leaving nanometric holes in the Au film. The walls and base of the holes are Au and glass respectively.

When these gold films are illuminated in a transmission mode with “white light”, i.e. perpendicular to the surface of the film, localized surface plasmon resonance in the vicinity of the nanoholes results in a resonance peak in the transmission spectrum. Typically the nanohole chip was illuminated with a fiber-optic probe illuminating an area of about 0.1 mm2. The position of the resonance peak of this transmitted light (at about 500 nm) has been found to be a function of the refractive index of the adjacent medium in the immediate vicinity of the holes. Using pure liquids with refractive indicies ranging from 1.333 to 1.478 a red-shift of approximately 110 nm per refractive index unit was observed.

Binding and adsorption of biomolecules to the gold and glass surfaces result in a red-shift of the localized surface plasmon resonance peak in the transmission spectrum of the gold holes. In order to evaluate the selectivity of the method, an antibody to the tumor marker CA 19-9. was immobilized to the surface resulting in a red-shift of about 10 nm. Exposure of the surface to BSA or another antigen, tumor marker (carcinoma embryonic antigen) did not result in further shifting of the peak. However exposure of the CA 19-9 antibody loaded chip to the CA 19-9 antigen resulted in an additional 3 nm red-shift. We estimate the sensitivity of the method for measuring the binding of proteins is at least 4x10-16 moles on the ~ 0.1 mm2 probing area.

Because of the simplicity of the fabrication method, and the small probing area in the transmission configuration, we expect that this technique could be developed into a simple, robust, and inexpensive platform for fabrication of a variety of label-free and array-based biosensors for high throughput screening of protein-protein interactions.

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