Recent Trends in the Application of Evanescent Wave Biosensors

Abstract

During the last few years biosensors have become important for the investigation of biomolecular interactions. In particular surface plasmon resonance (SPR) and resonant mirror (RM) techniques [1] are frequently used in biosensors. More than one thousand publications concerning the use of SPR or RM biosensors underline the importance of these techniques in the analysis of interactions between biomolecules.SPR and RM techniques are optical methods which use evanescent waves [1] to primarily determine the refractive index of an (aqueous) solution. As a consequence, these methods are unspecific, however they have the advantage of being very sensitive. Specificity can be introduced by modification of the surface facing the solution, for example, by immobilization of a receptor protein. Extremely thin layers of gold, other precious metals or glasslike materials may be used as supporting surfaces. Due to the interaction with the immobilized protein a bioactive molecule, present in solution above the sensor, will be attracted to the surface. The obtained change of the refractive index of the solution close to the sensor surface is directly proportional to the increase or decrease in mass of the sensor during the ligand ± receptor binding process (Figure 1).The experiments can be performed within minutes and need only very small amounts of sample, making them interesting as pharmaceutical screening processes. However, for biological aspects the signal progress over time is much more interesting, since one can determine the equilibrium and time constants (the association rate constant or ªon-rateº, and the dissociation rate constant or ªoff-rateº) of the ligand ± receptor binding process from the curves. Due to the optical detection mode, one obtains insights into the time scale of the processes being monitored without labeling the molecules. The collected kinetic and thermodynamic information complement structural data of the systems under investigation, thus being of great importance for structure activity relationship (SAR) studies of biomolecular complexes. Membrane Systems on Sensor SurfacesImmobilization of a molecule onto the sensor surface results in reduced translational and rotational degrees of freedom. This fact distinguishes SPR and RM techniques from other biophysical methods where all interacting partners are dissolved. However, many important biomolecular recognition processes take place on surfaces, such as cell membranes. The logical consequence is, therefore, to build artificial membranes on sensor surfaces to study receptor ± ligand interactions in an environment most closely resembling the natural one. To achieve this goal, different concepts have been developed to immobilize membranes and embedded proteins on sensor surfaces. The most apparent and simple one is to couple alkanes through a thiol group directly to the gold surface. These functionalized alkanes create a hydrophobic, self-assembling monolayer on the sensor surface, on which a lipid bilayer can be formed (Figure 2 a). The use of these me...

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