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Bulk and spikes
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Bulk and Spikes

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.

Fresh buffers

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.

Baseline drift

Baseline drift is usually a sign of non-optimal equilibrated sensor surfaces. Drift is often seen directly after docking a (new) sensor chip or after the immobilization of the sensor surface. This is due to the rehydration of the surface and the wash-out of chemicals used during the immobilization procedure or the adjustment of the bound ligand to the flow buffer. It can be necessary to run the running buffer overnight to equilibrate the surfaces.
Drift can also occur after a change in running buffer. In general, prime the system after each buffer change and wait for a stable baseline. Failing to equilibrate the system will result in a waviness pump stroke because the previous buffer is mixing with the next buffer in the pump. After some pump strokes the signal will be stable again.

Start-up drift can be seen when the flow is initiated after a flow stand still. Some sensor surfaces are susceptible to flow changes and this will be visible as a drift that will level out over time (5–30 minutes). The duration of this effect depends on the type of sensor and the ligand bound to it. It is advised to wait for a stable baseline before injection of the first sample. In systems where this is not possible, a short buffer injection and a five minute dissociation time can stabilize the baseline before the analyte injection is done. Regeneration solutions can also have an effect on the drift. This can be different between the reference and active surface due to the difference in protein and immobilization level. When using long dissociation times, equal drift rates between channels must be established or double referencing must compensate for drift differences sufficiently.
To equilibrate the system, use sufficient buffer in prime and wash steps. Flow the running buffer at the flow rate of the experiment until a stable baseline is obtained.

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 and 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).

Pump spikes

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.

Air bubbles

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.

Carry-over

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.

Sample separation

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.

Equilibration, baseline and noise level

Equilibration of the system is done by flowing running buffer over the sensor surfaces and monitoring the baselines. Start with preparing sufficient buffer for the experiment and filter and degas the solution. Add the appropriate detergent after filtering and degassing to avoid foam forming. Change the buffer in the pumps and tubing by priming the system several times or by flowing the buffer through the system.

To determine the noise level of the instrument, first equilibrate the system to minimize drift. Then inject running buffer several times and observe the average baseline response.
In the figure ‘Buffer injection’, a 5 minute equilibration time is set before the injection is started (1). When the injection needle hits the injection port (2), the baseline drops an average of 2 RU's. The pump fill (3) stops the flow for some seconds and a spike in the system occurs. At point (4), the buffer injection starts and it ends at point (5).

Injection and noise
Buffer injection

The system is very sensitive to pressure differences, which cause abrupt response changes. Nevertheless, the overall noise level is very low (< 1 RU). If one of the curves is used as a blank, the overall signal will be < 1 RU even during the buffer injection.

The average response level is the starting point for the next experiments. In addition, check the shape of the curves. If there is drift or the curves are not level shortly after the injection start, equilibrate better or clean the instrument. If the response between the flow channels is not comparable, it may be an indication that the IFC or sensor needs replacement (5) or that the detector should be recalibrated.

Therefore, it is advised to use a steady running buffer flow and incorporate several dummy injections (running buffer) with regeneration at the start of an experiment to stabilize the system. In addition, in systems where this is not possible, a short buffer injection and a five minute dissociation time can stabilize the baseline.

Double referencing

Double referencing is the procedure to compensate for drift, bulk effect and channel differences. First a reference (negative) channel is subtracted from the active channel. This will compensate for the main bulk effect and drift. Then the blanks (running buffer only) are subtracted. This will compensate for differences between the reference and active channel. To have the best referencing the reference channel should closely match the active channel. Use several blanks during the measurement and space the blanks evenly within the experiment.

Double referencing
Double referencing

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.

References

(1)BioNavis BioNavis website. (2020). 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
(5)Myszka, D. G. Improving biosensor analysis. J.Mol.Recognit. 12: 279-284; (1999). Goto reference