Non-specific binding

Non-specific binding can occur between the analyte and the negatively charged (dextran-)matrix (unreacted carboxyl groups) (1) or between the analyte and ligand. The source of the non-specific binding can be the analyte, a contaminant in the sample or the sample matrix. Non-specific binding is often hydrophobic in character and strong enough to withstand common regeneration conditions (2). SPR cannot differentiate between the binding of a specific analyte and the binding of a non-specific analyte. Therefore, take special care to recognize and avoid non-specific binding.

To investigate the influence of the surface matrix, use a native and a deactivated sensor chip surface. By simply injecting the analyte at the highest concentration over the two sensor chip surfaces, non-specific matrix binding becomes apparent. A minor binding by the analyte, may be compensated for using a reference surface (3). The use of smaller or less charged matrices may reduce non-specific binding. Deactivating the surface with other molecules like ethylenediamine, PEG amine, aspartate or glutamate can also help to reduce non-specific binding.

A change in buffer conditions, for instance, adding more salt (up to 0.5 M NaCl), a detergent (0.005%—0.05% P20) or chemicals (3 mM EDTA) can minimize non-specific binding. The addition of carboxyl methyl dextran (0.1—10 mg/ml) to the sample can reduce non-specific binding even further.

Although carboxylated dextran is the most commonly used sensor chip surface, it can suffer from a relative high level of fouling in bio fluids like plasma. Therefore, it can be beneficial to use alternative surface chemistries when using complex solutions (4),(5). Proposed coatings are alkanethiolates terminated with diethylene glycol and carboxylic groups, poly(ethylene glycol) grafted onto the SAMs and zwitterionic polymer brushes of poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), and poly(phosphorylcholine methacrylate) (6).

Reference surface

Use a reference surface (7),(8) to compensate for matrix effects, refractive index effects and non-specific binding of the analyte. It is important to match the reference surface with the other surfaces as close as possible. Depending on the nature of the ligand and analyte there are three types of reference cells.

• The unmodified sensor surface
• The deactivated sensor surface
• The matched sensor surface

Check the suitability of a reference sensor surface by injecting the analyte at the highest concentration that you expect to use. If the non-specific binding is low, use this surface.

Especially with low ligand densities it is important to match the reference cell as close as possible with the other cells. Try to use an inactive ligand or a similar protein like a non-related IgG or use BSA to mimic a protein surface.

By using blank injections (flow buffer only) and applying double referencing most of the differences between the reference and active channels can be compensated.

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 in the analyte solution. The difference in channel behaviour 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 (9). This will provide essential information about the reference surface versus the specific surfaces. When differences between the reference and specific surfaces are observed, a calibration plot has to be made (10).

To measure low molecular mass analytes, a system of four flow cells, each with a higher ligand concentration, can be used. By proper bulk subtraction even small response changes can be measured accurately (11).

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 (12). More detailed information can be found in (7).