David G Fernig 1 Introduction

Networks of interacting molecules, operating from the outside of the cell to the cell nucleus, regulate cell behavior. Optical biosensors provide a means of analyzing these interactions and possess key advantages over other methods: posttranslationally modified proteins, secondary gene products such as polysaccharides, chemically synthesized molecules, and nucleic acids are all equally susceptible to analysis; a quantitative description of an interaction is obtained; the structural rules and the kinetics governing the formation of multimolecular assemblies can be probed.

The principles of measurement underlying optical biosensors have been reviewed in detail elsewhere (1), and only a basic description will be given here. Optical biosensors consist of a sensor surface, on one side of which reside the optics that enable measurements to be made and on the other side of which resides the liquid phase (see Fig. 1). The optical system generates an exponentially-decaying evanescent wave that penetrates into the liquid phase. The greatest sensitivity occurs where the evanescent wave is strongest, that is, closest to the surface. In general, useful measurements can be made within about 200 nm of the surface. The optics essentially measure changes in refractive index, so the signal obtained depends on the refractive index within this 200 nm.

The essence of experiments using optical biosensors is that one partner of a molecular interaction is immobilized on the surface and the interaction of the other partner(s) is then followed in real time by adding them to the liquid phase. It is usual to refer to the immobilized molecule as the immobilized ligand or ligand and the soluble partner(s) as the soluble ligate(s) or ligate(s). The signal obtained from proteins and nucleic acids depends solely on the amount of material at the surface. Hence with these macromolecules, the optical biosensor acts as a very sensitive mass sensor. However, glycosaminoglycans have a low refractive index, which may vary with the degree of sulfation (Fernig, D. G., unpublished). Therefore, compared to proteins, glycosami-

From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Fig. 1. Schematic of an optical biosensor.

noglycans give a small signal on a mass basis, and this signal cannot be related directly to the amount of material, since it may depend on composition. For these reasons, experiments should always be designed such that the proteoglycan, its glycosami-noglycan chains or oligosaccharides derived from the latter are immobilized on the surface and the protein partner(s) are used as soluble ligate. Unfortunately, pro-teoglycans often contain more than one glycosaminoglycan chain, and each chain may contain more than one binding site for the protein partner of interest. Multivalent ligands usually have high avidity. Molecules with high avidity exacerbate the major artifacts encountered in optical biosensors (see Subheading 3.5.5.).

There are currently four commercial instruments on the market, BIAcore (Pharmacia Biosensor, Uppsala, Sweden), BIOS-1 (Artificial Sensing Instruments, Zurich, Switzerland), BioTul (BioTul, Munich, Germany), and IAsys (Affinity Sensors, Cambridge, UK). Two of these instruments, the BIAcore and the BioTul, use surface plasmon resonance to produce the evanescent wave in order to probe the liquid phase and thus have a gold film at the sensor surface. The ability of thiol groups to bond gold is used to modify the surface so as to make it amenable to the attachment of biological macromolecules. Available surfaces include carboxymethyl dextran for BIAcore and BioTul, and for BIAcore only, carboxymethyl dextran derivatized with streptavidin and nitrilotriacetic acid, short carboxymethyl dextran, dextran with a low degree of carboxylation, planar hydrophobic, carboxylated, and gold. In contrast, the BIOS-1 and IAsys use an evanescent wave generated by waveguiding to probe the liquid phase. The IAsys has a layer of metal oxide at the sensor surface, which enables molecular deposition of a variety of groups, and, in addition to carboxymethyl dext-ran, hydrophobic, amino, carboxyl, and biotin surfaces are available. There are two issues to bear in mind regarding the surfaces. First, they will possess holes at the atomic level and thus some gold or metal oxide may be exposed to the experiment. Second, carboxymethyl dextran gels possess hydrophobic pockets, which may either cause nonspecific binding artifacts or be a useful functionality.

The liquid phase, in which experiments take place (see Fig. 1), consists of two components, a homogenous bulk phase that is mixed by a vibrational stirrer (IAsys) or by flow (BioTul and BIAcore), and a thin stationary phase next to the sensor surface, whose constituents exchange by diffusion with the bulk phase. The ligand immobilized on the sensor surface is within the stationary phase. The depth of the stationary phase depends on the efficiency of mixing, which in turn determines the likelihood of diffusion artifacts (see Subheading 3.5.5.). The vibrational stirrer in IAsys should always be set to maximum. Since it induces chaotic mixing at the interface between the bulk phase and the stationary phase, the latter is reduced to a minimum and exchange between the two phases is efficient. In flow systems, mixing depends on the rate of flow (BioTul and BIAcore), and this should always be set to the maximum. The issue of mixing is compounded with laminar flow systems (BIAcore), since, as the bulk phase approaches the stationary phase, the rate of flow decreases, thus reducing the efficiency of mixing at the bulk phase-stationary phase interface.

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