7 - Small Molecule Interaction

7.1 General aspects

7.1.1 Ratio of solution sample vs. surface sample

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.

7.1.2 Calculation Example

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:

1000 RU Immob.-level: 2.5 RU
2000 RU Immob.-level: 5 RU
4000 RU Immob.-level: 10 RU
8000 RU Immob.-level: 20 RU

Rmax can also be calculated with equation 7-1 [https://www.sprpages.nl/sensorgram-tutorial/a-curve].

$Rmax_{analyte} = { {Mr_{analyte}\cdot R_{ligand}\cdot Valency_{ligand}}\over{Mr_{ligand}} }[RU]$

(7-1)

Note: The activity of the surface sample is often lower (≤80%), due to the used immobilization and/or purification procedures.

7.1.3 Solution sample with low solubility in aqueous solution

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.

7.1.4 Why is DMSO calibration needed?

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.

7.2 Experimental hints

7.2.1 Low binding responses expected

Please note:

Include reference surfaces
- needed to subtract non-specific binding of solution samples
Include enough zero injections
- needed to subtract bulk shifts and drifts
- needed to improve data quality
Test activity of surface sample with standard compound (if available)
- needed to track activity of the surface sample (target) over the whole experiment
- e.g. include every 10^th cycle an activity test cycle, consisting of a separate zero and a standard compound at a concentration 5 – 10 × K_D
- needed to adjust calculation of theoretical Rmax vs. experimental Rmax

7.2.2 Accurate sample preparation needed

Please note:

Prevent mismatch in DMSO content of running buffer and solution sample
- the less mismatch, the less to be corrected
- a difference of 1% DMSO from running buffer to sample a signal of ~1200 RU can be obtained
Example for sample preparation
- Stock of solution sample to be injected: 10 mM in 100% DMSO
- DMSO – content of the running buffer: 3% DMSO
- Preparation of solution sample stock: dilute the solution sample (10 mM in 100% DMSO) to 3% DMSO using running
  buffer, no DMSO (e.g.: 97 µL of running buffer, no DMSO + 3 µL of 10 mM Sample, 100% DMSO), this will lead to a stock of 300 µM of your sample, ~3% DMSO
Preparation of solution samples for the experiment
- dilute the solution sample stock to e.g. 20 µM using the running buffer, 3%
  DMSO
- all other dilution were made using the running buffer, 3% DMSO

7.2.3 Accurate DMSO calibration / solvent correction needed. Please note

Example for DMSO calibration curve needed for running buffer containing 3% DMSO:

Prepare your normal running buffer (e.g.: PBS with 0.05% Tween20)
Prepare the DMSO Buffer (e.g.: normal running buffer + 3% DMSO)
Further dilutions (e.g. 2.4% or 3.6%) should be prepared in the same manner as shown in the table 7-1

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]
2.6	133	867	/
2.8	67	933	/
3	/	1000	/
3.2	/	998	2
3.4	/	996	4

Injection Parameter for solvent correction:

Use same flowrate as used for solution sample
Use 20-40 s of contact time
Use “solv. Corr.” injection type
Calibration curve is automatically calculated/generated from analysis software
Example of a 5-Pnt. Calibration curve is shown in figure 7-2

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.