Experimental Setup

The most basic experimental setup for TIRE includes a glass slide with a thin metal film mounted on a prism and an ellipsometer. In addition, depending on the specific application and measurement conditions, components like a flow cell, a flow control system, application-specific data acquisition, and system control software are needed. These components can be universal, or application specific, depending on the user needs. The setup can be simplified to an absolute minimum if a sensor-type measurement tool is of interest, or extended with various additional components if complex analyses are in focus. It is impossible to give review of all possible experimental setups for TIRE, due to the continuous development and variety of possible setups. In the following paragraphs we try to give a general overview of the TIRE components of importance for protein studies, focusing on possible advantages and disadvantages of particular solutions. Examples of application-specific setups can be found later in this chapter. The suggestions and opinions presented here should be treated as comments and they certainly do not cover all possible aspects of the presented setups. One should notice that when a specific application is of interest, particular component types may depend on each other, and understanding of the right choice for the best possible results may not be straightforward.

The most specific part of the TIRE setup is a glass slide sample coupled to a glass prism. The samples maybe the objectives for investigation by TIRE, or may serve as surfaces for additional treatments. In the case of protein adsorption studies, semitransparent metal layers are used. In principle, the metal layer can be deposited directly onto the prism surface, but in most cases it is deposited onto a glass slide, which then is mounted in optical contact with the prism. The glass slide should have the same optical properties as the prism. To avoid unnecessary interference from the prismslide interface an index-matching liquid should be used. If TIRE should be extended with SPR, a thin layer of suitable metal (preferable gold, silver, or copper for the best effect) should be used on the glass slide. The thickness of the layer depends on the application, and again, in the case of SPR-enhanced TIRE it should be tuned to the specific application, prism type, and the wavelength range available on the ellipsometer. An example of a setup with an equilateral prism is presented in Fig. 4.

One of several possible prism geometries that can be used in TIRE is a triangular equilateral prism, which allows measurements at an angle of incidence of 60°. Other values for the angle of incidence are also possible with a 60° prism. However, one must then take into account the refraction of the incoming and outgoing light at the two prism-air interfaces, as the light is not incident normal to the prism surface. To obtain the best results,

Prism

Fig. 4. Example of the prism and sample configuration for TIRE. Notice the index-matching liquid inserted between the prism and the glass slide and the metal layer deposited on the glass slide

a prism that is suitable for the specific application should be used. Again, in the case of protein adsorption studies, when the SPR effect is utilized, one should use a triangular (or for practical reasons trapezoidal, as the height of the prism increases with internal angle) prism with its internal angle close to the angle of incidence at which the SPR effect occurs for the selected wavelength, metal layer thickness, and material composition. To allow multiple angles of incidence one can use a prism with a polygonal shape, or a prism that together with the glass slide forms a half-cylindrical configuration; this is important to notice, as otherwise the light will be refracted on the air-glass interface, resulting in a different internal and external angle of incidence. With a half-cylindrical configuration, one also needs to remember that the incident parallel light beam from the ellip-someter has a certain, finite width and thus it will be focused at the side of the prism onto which the glass slide is mounted. This can minimize the sensing area, but also introduce unwanted effects due to the refraction of the peripheral parts of the beam on the prism surface. To avoid this, the light should be prefocused before entering the prism so that the whole beam is incident normal to the prism surface.

To be able to perform in situ studies, for example for monitoring the adsorption of proteins onto the surface, the prism and the glass slide with the metal layer have to be mounted in an appropriate flow cell. Virtually any type of flow cell can be used in a TIRE setup, although in most cases an application-specific design is preferred. In Fig. 5, an example of an injection-type flow cell is presented. In this type of cell the liquid is injected close to the sample surface and then pumped out from the top of the cell. The flow rate as well as solution mixing can be controlled by a computer-

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