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Solvent correction

The need for solvent correction procedures arises because subtraction of the reference response does not exactly eliminate the contribution of bulk solution to the measured response. Bulk solution is excluded from the volume occupied by ligand molecules on the ligand spot, so the bulk contribution to the relative response is smaller than on the reference spot. The difference is small (usually less than 10-20 RU), but it can be of the same order of magnitude as the responses expected from binding of low molecular weight lead compounds.

Bulk solution is excluded from the volume occupied by ligand molecules on the ligand spot, so the bulk contribution to the relative response is smaller than on the reference spot.

As long as buffer of constant composition flows over the sensor surface, the excluded volume effect introduces a constant error in reference subtraction which may be ignored for practical purposes.

The same is true during sample injection if the refractive index of the sample differs by a constant amount that of the running buffer, since in that case the error will be the same for all samples and the relative response will still be valid. However, if the difference between sample and running buffer varies between different samples, the excluded volume effect will introduce different errors in different samples so that a comparison between samples will no longer be correct.

Samples dissolved in buffer containing DMSO for solubility reasons are particularly susceptible to this effect. Organic solvents like DMSO contribute very significantly to the refractive index of the bulk solution, and small variations in DMSO concentrations between samples can introduce relatively large shifts in the bulk response, with corresponding errors in relative response values. The solvent correction procedure takes care of this situation.

When is solvent correction necessary?

Solvent correction is necessary under a combination of the following circumstances, commonly met in work with low molecular weight analytes such as drug candidates:

• The samples and running buffer contain DMSO
• The analyte is a small molecule and expected responses are low
• The ligand is a macromolecule immobilized at a high density on the surface
• The bulk response is high in relation to the measured binding response and is subject to variations between sample solutions

The combination of these three factors results in variations in the magnitude of the excluded volume effect that are significant in relation to the measured analyte responses, so that samples with expected bulk contributions cannot be referenced properly.

In drug discovery and development work, the analytes are often small molecules which give correspondingly low response values (typically of the order of 50-100 RU or less). High levels of immobilized protein ligands (several thousand RU) are used to maximize the analyte response. Addition of DMSO to samples and buffers gives a high bulk response. A difference of 1% in DMSO concentration corresponds to a difference of about 1200 RU in bulk response, so that small variations in DMSO concentration, unavoidable in the preparation of a large number of diverse samples, lead to significant differences in bulk response between samples. Solvent correction should generally be included in assay protocols for this kind of work: the decision as to whether to apply the solvent correction or not can be when the results are evaluated.

If you are running assays with macromolecular analytes, the expected responses will be higher and the level of immobilized ligand usually lower than for small molecules, and inclusion of DMSO in buffers and samples is seldom necessary. Solvent correction should be omitted in such cases.

See also

List of help files

Solvent correction for different applications

Solvent correction procedure

Assessing solvent correction curves