Recognising problems in your experimental setup is important. Baseline drift is usually a sign of not optimal equilibrated sensor surface. It is sometimes necessary to run the flow buffer over night to equilibrate. Several buffer injections before the actual experiment can minimize drift during analyte injection.
Avoid bulk shifts at the beginning and end of the injection by matching flow and analyte buffer. Low shifts (< 10 RU) due to buffer differences are easy compensated for by the reference surface but avoid larger. Sudden 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 viscosity solutions.
When the response during the analyte injection is dropping it can indicate that there is sample dispersion. The sample is mixing with the flow buffer, resulting in an effective lower analyte concentration (5). Most SPR machines have special routines to separate the flow buffer from the sample. Use them and check if the sample is properly separated from the flow buffer.
Problems with carry over and sample dispersion are easily recognised by injecting an elevated NaCl solution (0.5 M) and a flow buffer solution. The NaCl solution must give a sharp rise and fall when injected have a flat steady state. The flow buffer injection should give an almost flat line indicating that the needle was sufficient washed.
A negative signal with SPR
Sometimes a negative binding signal is seen in an SPR experiment. It is not always obvious where this negative binding signal comes from. Below is a list of possible causes and possible solutions.
Some negative binding responses originate from buffer mismatches. In general a low ionic analyte solution will give a negative jump compared to the flow buffer. In general, a buffer mismatch of 1 mM NaCl will give a jump of 20 RU in a Biacore-type instrument on a CM5 sensor chip.
Furthermore, the dilution of the analyte in the flow buffer may alter the total salt content. In addition the analyte itself may have an influence on the behaviour of the SPR-measurement.
The buffer mismatch can be eliminated by dialysing the analyte. However, if the problem persists and is proportional to the analyte concentration, it is most likely originating from the analyte itself.
Some negative binding responses originate from difference between the reaction of the reference and the reaction of the ligand channel to the injected analyte solution (buffer composition, additives, pH or analyte concentration). The difference in reaction is explained by the variation in ligand density between the reference and ligand channel. When immobilizing, the actual amount of ligand on the sensor surfaces differs between the sensor surfaces. This difference is not only in response units or in molarity, but also in the volume the ligand is occupying in the matrix. When the flow buffer is replaced by the analyte (buffer), the matrix can swell or shrink depending on the difference. Because of the different ligand volumes in the matrices, the volume exclusion of the buffer is different; hence the reaction to the buffer changes (2),(3).
In situations with for instance DMSO or glycerol in the analyte buffer, differences can be relatively large and result - after reference subtraction or double referencing - in negative responses in the original curves. In these cases it can be beneficial to make a calibration plot to compensate for the volume exclusion (1),(3).
Non-specific matrix interaction
Some negative binding responses originate from the interaction of the analyte and sensor chip matrix. Especially the dextran based sensor chip can have high affinity for small compounds. Adding CM-dextran (0.1 - 1 mg/ml) to the flow buffer can lower the non-specific interaction with the dextran matrix. Other solutions are to use sensor chips with no dextran or other types of linkers, like alginate (Bio-Rad) or dendritic polyglycerol (Xantec). It is even possible to make your own sensor chip surface. In this way you can add a SAM to the sensor gold layer that will suit you best (4).
Non-specific reference interaction
Some negative binding responses originate from non-specific binding of the analyte to the ligand on the reference channel. This should be easy to detect by overlaying the raw data of the reference channel and ligand channel. From the sensorgram you can sometimes deduce if this is a strong or weak non-specific interaction (observe the dissociation). If the interaction seems to be weak, adding extra detergent (e.g. 0.02%), extra salt (e.g. 250 mM NaCl) or changing the pH can suppress this interaction. Adding BSA (0.1 – 1 mg/ml) or CM-dextran (0.1 – 1 mg/ml) to the flow buffer can also lower the non-specific interaction. In addition, reversing the interaction system can solve this problem.
In all situations, the reference surface is crucial. The easiest surface is one that is native and thus unmodified. The dextran-based sensor chips (e.g. BIACORE CM5) contain a lot of carboxyl groups (-COOH) which become negatively charged at pH ~7. Activating (NHS/EDC) and deactivating with ethanolamine replaces the carboxyl groups with a hydroxyl group (-OH), and hydroxyl groups are less negatively charged at a physiological pH.
Immobilizing the reference channel with a non-related ligand (e.g. protein, oligo) is not always straight-forward. Often BSA is chosen because it is readily available and pure. However, BSA can bind a lot of molecules and should not be the first choice. In addition, albumin (when ionized in water at pH 7.4, as found in the body) is negatively charged. Other molecules that can be used include, for instance, non-related antibodies (IgG). Most laboratories have IgG for detection of proteins of interest and one can choose an antibody that is not binding to the analyte of interest and use it as a reference surface.
Determining the correct amount of ligand to be immobilized as a reference surface is the next difficult part. Start with immobilizing an equal number of response units compared to the ligand of interest. This will give a similar buffer volume exclusion compared to the ligand of interest. Only testing and comparing will show if you have to immobilize less or more.
Solving negative curves
Steps you can take to test the suitability of your reference channel (you can make a dedicated chip for this):
- Inject the analyte at the highest concentration over a native surface
- Inject the analyte at the highest concentration over a deactivated surface
- Inject the analyte at the highest concentration over a BSA or IgG surface
Steps you can take to reduce non-specific binding and lower difference between reference and ligand channel
- Add extra detergent or salt to the flow buffer
- Add BSA or CM-dextran to the flow buffer
- Add blank injections for double referencing
- Add calibration injections and make a calibration plot to correct for volume exclusion effects
In addition, consider reversing the interaction system and different sensor chip matrices.
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|(2)||Karlsson, R. Real-time competitive kinetic analysis of interactions between low-molecular-weight ligands in solution and surface-immobilized receptors. Analytical Biochemistry 221: 142-151; (1994). Goto reference|
|(3)||Karlsson, R., M. Kullman-Magnusson, M. D. inen, et al. Biosensor Analysis of Drug-Target Interactions: Direct and Competitive Binding Assays for Investigation of Interactions between Thrombin and Thrombin Inhibitors. Analytical Biochemistry 278: 1-13; (2000). Goto reference|
|(4)||Metzger, J., P. von Landenberg, M. Kehrel, et al. Biosensor analysis of beta2-glycoprotein I-reactive autoantibodies: evidence for isotype-specific binding and differentiation of pathogenic from infection-induced antibodies. clin. chem. 53: 1137-43; (2007). Goto reference|
|(5)||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|