Reference surface

Use a reference surface (1),(2) to compensate for matrix effects, refractive index effects and non-specific binding of the analyte. It is important to match the reference surface as close as possible with the other surfaces. Just activation and deactivation is not the ideal control. Try to use an inactive ligand or a similar protein like a non-related IgG or use BSA to mimic a protein surface.

It is possible due to differences in ligand density and immobilization that both reference and active surface react differently to changes in ionic strength or organic solvents like DMSO. This is caused by the different displaced volumes and ligand properties. This type of artefact can be detected by injecting a control solution with the same refractive index as the analyte solution (3). This will give essential information about the reference surface versus the specific surfaces. When differences between reference and specific surfaces are observed, a calibration plot has to be made (4).

To measure low molecular mass analytes, a system of four flow cells with each a higher ligand concentration can be used. By proper bulk subtraction even small response changes can be measured accurately (5).

Even with a reference surface, it is better to eliminate bulk and drift effects. Carefully designed procedures and matched buffers will enhance the data fitting process. In any case, always use pure, filtered and degassed solutions (6). More detailed information can be found in (2).