16-05-2012, 12:21 PM
Surface plasmon resonance
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1. Introduction
Since the development almost a decade ago (1,2) of the first biosensor based on surface plasmon resonance (SPR), the use of this technique has increased steadily. Although there are several SPR-based systems (3-5), by far the most widely used one is the BIAcore (1,2), produced by BIAcore AB, which has developed into a range of instruments (Table 1). By December 1998 over 1200 publications had reported results obtained using the BIAcore. It is likely that it would be even more widely used were it not for its high cost and the pitfalls associated with obtaining accurate quantitative data (5-8). The latter has discouraged many investigators and led to the perception that the technique may be flawed. This is unjustified because the pitfalls are common to many binding techniques and, once understood, they are easily avoided (4,8,9). Furthermore, the BIAcore offers particular advantages for analysing weak macromolecular interactions, allowing measurements that are not possible using any other technique (4,10). This Chapter aims to provide guidance to users of SPR, with an emphasis on avoiding pitfalls. No attempt is made to describe the routine operation and maintenance of the BIAcore, as this is comprehensively described in the BIAcore instrument manual
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(see Section 9.3). Although written for BIAcore users, the general principles will be applicable to experiments on any SPR instrument.
2. Principles and applications of Surface Plasmon Resonance.
2.1. Principles
The underlying physical principles of SPR are complex (see Section 9.1). Fortunately, an adequate working knowledge of the technique does not require a detailed theoretical understanding. It suffices to know that SPR-based instruments use an optical method to measure the refractive index near (within ~300 nm) a sensor surface. In the BIAcore this surface forms the floor of a small flow cell, 20-60 nL in volume (Table 1), through which an aqueous solution (henceforth called the running buffer) passes under continuous flow (1-100 μL.min-1). In order to detect an interaction one molecule (the ligand) is immobilised onto the sensor surface. Its binding partner (the analyte) is injected in aqueous solution (sample buffer) through the flow cell, also under continuous flow. As the analyte binds to the ligand the accumulation of protein on the surface results in an increase in the refractive index. This change in refractive index is measured in real time, and the result plotted as response or resonance units (RUs) versus time (a sensorgram). Importantly, a response (background response) will also be generated if there is a difference in the refractive indices of the running and sample buffers. This background response must be subtracted from the sensorgram to obtain the actual binding response. The background response is recorded by injecting the analyte through a control or reference flow cell, which has no ligand or an irrelevant ligand immobilized to the sensor surface.
One RU represents the binding of approximately 1 pg protein/mm2. In practise >50 pg/mm2 of analyte binding is needed. Because is it very difficult to immobilise a sufficiently high density of ligand onto a surface to achieve this level of analyte binding, BIAcore have developed sensor
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surfaces with a 100-200 nm thick carboxymethylated dextran matrix attached. By effectively adding a third dimension to the surface, much higher levels of ligand immobilisation are possible. However, having very high levels of ligand has two important drawbacks. Firstly, with such a high ligand density the rate at which the surface binds the analyte may exceed the rate at which the analyte can be delivered to the surface (the latter is referred to as mass transport). In this situation, mass transport becomes the rate-limiting step. Consequently, the measured association rate constant (kon) is slower than the true kon (see Section 7.2). A second, related problem is that, following dissociation of the analyte, it can rebind to the unoccupied ligand before diffusing out of the matrix and being washed from the flow cell. Consequently, the measured dissociation rate constant (apparent koff) is slower than the true koff (see Section 7.2). Although the dextran matrix may exaggerate these kinetic artefacts (mass transport limitations and re-binding) they can affect all surface-binding techniques .
2.2. Applications
This section outlines the applications for which SPR is particularly well suited. Also described are some applications for which it is probably not the technique of choice. Of course, future technical improvements are likely to extend the range of applications for which the SPR is useful.
2.2.1. What SPR is good for
2.2.1.1. Evaluation of macromolecules
Most laboratories studying biological problems at the molecular or cellular level need to produce recombinant proteins. It is important to be able to show that the recombinant protein has the same structure as its native counterpart. With the possible exception of enzymes, this is most easily done by confirming that the protein binds its natural ligands. Because such interactions involve multiple residues, which are usually far apart in the primary amino acid sequence, they require a correctly folded protein. In the absence of natural ligands monoclonal antibodies (mAbs) that are known to
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bind to the native protein are an excellent means of assessing the structural integrity of the recombinant protein. The BIAcore is particularly well suited to evaluating the binding of recombinant proteins to natural ligands and mAbs. Setting up an assay for any particular protein is very fast, and the data provided are highly informative.
2.2.1.2. Equilibrium measurements (affinity and enthalpy).
Equilibrium analysis requires multiple sequential injections of analyte at different concentrations (and at different temperatures). Because this is very time-consuming it is only practical to perform equilibrium analysis on interactions that attain equilibrium within about 30 min. The time it takes to reach equilibrium is determined primarily by the dissociation rate constant or koff; a useful rule of thumb is that an interaction should reach 99% of the equilibrium level within 4.6/koff seconds. High affinity interactions (KD < 10 nM) usually have very slow koff values and are therefore unsuitable for equilibrium analysis. Conversely, very weak interactions (KD >100 μM) are easily studied. The small sample volumes required for BIAcore injections (<20 μL) make it feasible to inject the very high concentrations (>500 μM) of protein required to saturate low affinity interactions (11).
Equilibrium affinity measurements on the BIAcore are highly reproducible. This feature and the very precise temperature control makes it possible to estimate binding enthalpy by van’t Hoff analysis (12). This involves measuring the (often small) change in affinity with temperature (Section 7.4). Although not as rigorous as calorimetry, much less protein is required.
2.2.1.3. Kinetic measurements
The fact that the BIAcore generates real-time binding data makes it well suited to the analysis of binding kinetics. There are, however, important limitations to kinetic analysis. Largely because of mass transport limitations it is difficult to measure accurately kon values faster than about 106 M-1s-1. This upper limit is dependent on the size of the analyte. Faster kon values can be measured with
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analytes with a greater molecular mass. This is because the larger signal produced by a large analyte allows the experiment to be performed at lower ligand densities, and lower ligand densities require lower rates of mass transport. For different reasons measuring koff values slower than 10-5 s-1 or faster than ~1 s-1 is difficult. Because the BIAcore is easy to use and the analysis software is user-friendly, it is deceptively easy to generate kinetic data. However obtaining accurate kinetic data is a very demanding and time-consuming task, and requires a thorough understanding of binding kinetics and the potential sources of artefact (Section 7.2).
2.2.1.4. Analysis of mutant proteins
It is possible using BIAcore to visualise the capture of proteins from crude mixtures onto the sensor surface. This is very convenient for analysing mutants generated by site-directed mutagenesis (13,14). Mutants can be expressed as tagged proteins by transient transfection and then captured from crude tissue culture supernatant using an antibody to the tag, thus effectively purifying the mutant protein on the sensor surface. It is then simple to evaluate the effect of the mutation on the binding properties (affinity, kinetics, and even thermodynamics) of the immobilised protein. This provides the only practical way of quantifying the effect of mutations on the thermodynamics and kinetics of weak protein/ligand interactions (15).
2.2.2. What SPR is not good for.
2.2.2.1. High throughput assays.
The fact the BIAcore can only sample can be analysed at a time, with each analysis taking 5-15 min, means that it is neither practical nor efficient for high throughput assays. Automation does not solve this problem because the sensor surface deteriorates over time and with re-use. Blockages or air bubbles in the microfluidic system are also common in long experiments, especially when many samples are injected.
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2.2.2.2. Concentration assays.
The BIAcore is also unsuitable for concentration measurements, because these require the analysis of many samples in parallel, including the standard curve. A second problem is that, for optimal sensitivity, concentration assays require long equilibration periods.
2.2.2.3. Studying small analytes.
Because the SPR measures the mass of material binding to the sensor surface, very small analytes (Mr <1000) give very small responses. The recent improvements in signal to noise ratio have made it possible to measure binding of such small analytes. However a very high surface concentration of active immobilised ligand (~1 mM) is needed, and this is difficult to achieve. Furthermore, at such high ligand densities accurate kinetic analysis is not possible because of mass-transport limitations and re-binding (Section 7.2). Thus only equilibrium analysis is possible with very small analytes, and then only under optimal conditions. This assessment may need to be revised as and when future improvements are made in the signal to noise ratio.
3. General principles of BIAcore experiments
3.1. A typical experiment
A typical SPR experiment involves several discrete tasks.
• Prepare ligand and analyte.
• Select and insert a suitable sensor chip.
• Immobilise the ligand and a control ligand to sensor surfaces.
• Inject analyte and a control analyte over sensor surfaces and record response.
• Regenerate surfaces if necessary.
• Analyse data.
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While the ligand and analyte could be almost any type of molecule, they are usually both proteins. This chapter focuses on the analysis of protein-protein interactions.
3.2. Preparation of materials and buffers
BIAcore experiments are frequently disrupted by small air bubbles or other particles passing through the flow system. Usually these can be flushed out and the experiment repeated, wasting only time and reagents. Occasionally, however, the damage is irreversible necessitating the replacement of an expensive sensor chip or integrated fluidic cartridge. This can be minimised by following a few simple rules (Section 9.2.1).
3.3. Monitoring the Dips
The output from the photo-detector array that is used to determine the surface plasmon resonance angle (θspr, Section 9.1) can be viewed directly as 'dips'. The current BIAcore documentation does not describe how to view and interpret dips, and so this information is supplied here (Protocol 1).
Protocol 1. Normal and abnormal 'dips'
Viewing the dips
1. Enter the service mode on the BIAcore control software by simultaneously pressing the control, alt, and s keys.
2. When the dialog box appears requesting a password ignore this and press the enter or return key. An additional Service menu will appear on the menu bar.
3. Select View dips from this menu.
4. A graph appears similar to the one in Figure 1 showing the amplitude of light reflected off the sensor surface (Reflectance) measured over a small range of angles. The angle of the minimum reflectance ( θspr) is calculated by fitting a curve to all this data, thereby enabling θspr to be measured at a far greater resolution than the resolution at which the data are actually collected.
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Normal dip
The important feature of a normal dip is its depth (Figure 1, Dip1). Generally it bottoms out at a Reflectance of ~10000. When the refractive index above the sensor surface increases (e.g. because of protein binding), the dip shifts to the right while maintaining its shape and depth and (Dip 2).
Abnormal dip
There are two main types of abnormal dip.
1. The shallow dip (Dip 3). The θspr is measured along a section of the sensor surface rather than at a single point. Refractive index heterogeneity on the surface gives heterogenous θspr values. When averaged the result is a shallow dip that does not reach below 10000 RUs. Heterogeneity can be the result of differences in the amount of material immobilised along the surface, in which case the dip is usually slightly shallow, or the result of small air bubbles or particles in the flow-cell, in which case the dip is very shallow.
2. No dip (Dip 4). A large change in the refractive index beyond the instrument dynamic range (Table 1) will shift the θspr so much that no dip is evident. Usually this is the result of air in the flow cell.
While slight shallowness of the dip is acceptable, and is common after coupling large amounts of protein, more severe abnormalities should not be ignored. Attempts should be made to return dips to normal by flushing the flow cells with buffer and/or regenerating the sensor surfaces. If this is ineffective a new sensor surface should be used.