Within the investigation of small molecules, the ratio of solution sample (analyte) vs. surface sample (ligand) is low. In detail: To measure low molecular weight compounds (≥100 Da) a high density of the surface sample is required.
When investigating the interaction between a surface sample (MW: 60 kDa) and a solution sample (MW: 150 Da) the theoretical Rmax, expecting 100% activity would be as follows:
Rmax can also be calculated with equation 7-1 [https://www.sprpages.nl/sensorgram-tutorial/a-curve].
Note: The activity of the surface sample is often lower (≤80%), due to the used immobilization and/or purification procedures.
As most low molecular weight compounds have a low solubility in aqueous solutions, these samples are often dissolved in 100% DMSO. In order to maintain the solubility of solution samples a certain amount of DMSO is needed in running buffer. Commonly used DMSO levels in the running buffers range from 1-5% of DMSO. Any mismatch in the DMSO content of the solution sample and running buffer will lead to high bulk shifts. Thus a DMSO calibration is required.
The SPR-signal is sensitive to refractive index changes near the sensor surface. If the refractive index of the injected solution sample is different from the running buffer an additional signal appears, the bulk response.
The bulk response is removed by subtracting the control surface from the active surface. This will work in the case of small bulk responses (~100 RU).
During the investigation of small molecule interactions using DMSO containing running buffer the subtraction of control surface from active surface is insufficient and will not eliminate the impact of the solvent to the response, because the reference surface will show a higher DMSO bulk response.
In detail more buffer molecules get replaced by DMSO molecules close to the reference sensor surface, if DMSO containing samples are injected. As the immobilized surface sample (ligand) occupies more space on the active sensor surface, less buffer molecules get replaced during a DMSO containing injection. Thus, the response coming from the reference surface is higher compared to the active surface. Such bulk response differences will be subtracted with the usage of a DMSO calibration. The DMSO calibration is explained in more detail in section 7.2.3.
Example for DMSO calibration curve needed for running buffer containing 3% DMSO:
Table 7-1: Pipetting scheme for DMSO calibration curve (1 mL per dilution)
|% of DMSO||Buffer, no DMSO [µL]||Buffer, with 3% DMSO [µL]||100 % DMSO [µL]|
Injection Parameter for solvent correction:
As shown in Figure 7-2, for each sample/injection that needs to be corrected, the bulk response from the reference surface will be used and result in a dedicated correction factor, that is subtracted from the individual injection.