Recognising problems in the experimental setup is important. Drift, jumps and spikes in the curves make analysing sensorgrams difficult. Analysing suboptimal sensorgrams will lead to erroneous results and waste experimental time. Therefore it is vital to identify problems in the experimental set-up and solve them before analysing is attempted. This part will high light several problems and give some hints to diagnose where the problem is and provide solutions to optimize the experiment.
Ideally fresh buffers are prepared each day and 0.22 µM filtered and degassed before use. We normally make 2 litres of buffer and 0.22 µM filter. Storage is in clean (sterile) bottles at room temperature. Keep in mind that buffers stored at 4°C contain more dissolved air which can create air-spikes in the sensorgram. Just before use, transfer an aliquot of the buffer to a new clean bottle and degass. It is bad practice to add fresh buffer to the old since all kind of nasty things can happen / growing in the old buffer. After degassing, add a detergent when suitable. A good running buffer hygiene is step one for better results.
Bulk shift jumps
Most analytes are not prepared with SPR in mind or need different formulations (buffers) to keep them happy in solution and storage. For instance many compounds are dissolved in DMSO and proteins are stored in glycerol. These solutions have a high refractive index compared to the standard running buffer and will cause high buffer jumps which can obscure the kinetics.
The main cause of bulk shift jumps is when running and analyte buffer are not matched. Non-matched buffers wil result in a shift of the curve at the beginning and end of the injection. Low shifts (< 10 RU) due to small buffer differences are easily compensated for by the reference surface but avoid larger bulk refractive index shifts.
If the analyte is provided in a different solution it can be beneficial to dialyse the analyte in the running buffer. As an alternative, buffer exchange of small volumes of analyte can be done with size exclusion columns.
If for instance DMSO is necessary to keep the analyte in solution, dialyse against the buffer with DMSO and use the solution of the last dialysis buffer exchange as running and dilution buffer. This will minimize buffer jump, since even small differences in DMSO concentration will result in large jumps in the sensorgram. Furthermore, cap the vials since evaporation of the analyte solution will also lead to buffer jumps during the analyte injection. The SPR instruments of BioNavis™ (1) can compensate for the bulk shift in real time with the function PureKinetics™ which measures the bulk refractive index of the solution. This allows having DMSO in samples and not in the running buffer.
Exluded volume differences
It is possible due to differences in ligand density after immobilization that both reference and active surface react differently to changes in ionic strength or organic solvents such as DMSO in the analyte solution. The difference in channel behaviour is caused by the different displaced volumes (excluded volume effect) and ligand properties. This type of artefact can be detected by injecting a control solution with the same refractive index as the analyte solution (2). 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 can be made to compensate for excluded volume differences (3).
When a system has to refill the pumps that deliver the running buffer, the flow rate will drop to zero for a short period of time. Since the flow stops there is a change in pressure and that can be seen as small spikes in the sensorgram.
In the same way, washing steps will result in a distortion of the curves. Special commands can be used to delay washing after analyte injection in order to have an undisturbed dissociation. In addition, care must be taken with placing report points since they should not overlap with a pump refill or washing sequences.
Another cause of spikes can be the build-up of small air bubbles in the flow channels. Especially at low flow rates (< 10 μ/min) the small air bubbles are not driven out fast enough, giving them time to grow until they come visible in the sensorgram. In addition, at high temperatures, e.g. 37°C air bubbles are easily formed. Thoroughly degassed buffers will help to minimize the formation of air bubbles. Keep in mind that when the instrument is fitted with a degasser, the running buffer is degassed but the sample not. High flow rates can also be used to flush out small air bubbles between cycles.
Sudden buffer jumps or spikes at the beginning of the analyte injection can point to carry-over. If this is observed, add extra wash steps between the injections. Take special care with high salt or high viscous solutions.
Ligand and analyte
Besides the buffer, the ligand and analyte can also be a source of distortions in the curve. Proteins are generally stored frozen and thawed shortly before use and possibly stored on ice. It is a good habit to mix en centrifuge the ligand and analyte for 10’ at 16000g before use to remove any aggregates.
As mentioned above, the analyte may differ in buffer composition compared to the running buffer. A high analyte dilution will lower the difference but cannot take it away totally. Keep in mind that the analyte itself can generate some bulk effect which will be larger at higher concentrations.
When the response during the analyte injection is dropping, it can indicate that there is sample dispersion. The sample is mixing with the running buffer, resulting in an effective lower analyte concentration (4). Most SPR instruments have a possibility to add one or more air bubbles between the sample and running buffer to separate the running buffer from the sample. Use the best injection routine and check if the sample is properly separated from the running buffer.
Spikes after reference subtraction
When reference and active channels with large bulk jumps are processed, spikes can be seen at the beginning and end of an injection. The overall association and dissociation curves are correct except at the beginning (1–4 seconds) and at the end of the injection. This is because the flow channels are in series and the arrival of the sample at the channel deviates slightly. The effect is that the reference is a little "out of phase" with the other channels. The spikes appear to be larger when the bulk effect is larger. Proper alignment of the curves can minimize these spikes. Some instruments offer inline reference subtraction which can minimizes this effect. In addition, changing all the samples to the running buffer can minimize the bulk refractive index problems further.
Injection system testing
After equilibration, check the injection system with several buffer injections and closely monitor the curves. For the best testing, use a new chip (plain gold or dextran coated). Make a solution with 50 mM extra NaCl to the running buffer. Then make a dilution series (e.g. 50, 25, 12.5, 6.3, 3.1, 1.6, 0.8, 0) and inject from low to high concentration (single cycle kinetics) and end with an injection of running buffer alone. This gives insight in how the system reacts to buffer differences and how the curves look like.
The highest NaCl concentration will be over 550 RU which shows that every 1 mM of salt gives on average a 10 RU bulk difference. Pay extra attention to the start and end of the injection. The rise and fall of the curve should be smooth and immediately and the steady state part should be even without drift up or down. The last running buffer injection serves as an indication if there is any carry-over from the previous injection.
In addition, inject the maximal volume to be sure that the analyte is properly separated from the running buffer.
|(1)||BioNavis - BioNavis website. (2022). Goto reference|
|(2)||van der Merwe, P. A. - Surface Plasmon Resonance. (2003).|
|(3)||Roos, H., R. Karlsson and K. Andersson - A calibration routine to improve the interpretation of low signal levels and low affinity interactions. (1998).|
|(4)||Rich, R. L. and D. G. Myszka - Survey of the year 2001 commercial optical biosensor literature. J.Mol.Recognit. 15: 352-376; (2002). Goto reference|
- Also read the Baseline drift topic.