Reference coupling

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).


(1) Dorn, I. T. et al Orientation and two-dimensional organization of proteins at chelator lipid interfaces. Biological Chemistry Hoppe-Seyler 379: 1151-1159; (1998).
(2) Ober, R. J. and Ward, E. S. The Choice of Reference Cell in the Analysis of Kinetic Data Using BIAcore. Analytical Biochemistry 271: 70-80; (1999).
(3) van der Merwe, P. A. Surface Plasmon Resonance. PDF file from internet (2003).
(4) Roos, H. et al A calibration routine to improve the interpretation of low signal levels and low affinity interactions. (1998).
(5) Karlsson, R. and Stahlberg, R. Surface plasmon resonance detection and multispot sensing for direct monitoring of interactions involving low-molecular-weight analytes and for determination of low affinities. Analytical Biochemistry 228: 274-280; (1995).
(6) BIACORE AB Kinetic and Affinity analysis using BIA - Level 2. (1998).