Products | SPR Biosensors | Applications

Introduction: Flexible SPR
We understand that not everyone is excited about Surface Plasmon Resonance (SPR) as we are, and so we are often asked: what can I do with SPR? | more...

Part I: Traditional SPR and XanTec
Kinetics | Thermodynamics | Concentration | Fragment screening | Determining selectivity | Determining specificity | more...

Part II: XanTec – Above and beyond
Quartz/Microscope flow cells | Photo-reactivity | Organic solid-phase reactions | Metal-organic frameworks | Coupling techniques (HPLC, mass spectrometry etc) | more...

Introduction: Flexible SPR

We understand that not everyone is excited about Surface Plasmon Resonance (SPR) as we are, and so we are often asked: what can I do with SPR?

We could simply answer 'almost everything', but that doesn't really help anyone reading this page. So instead, let's take a look at some different experiments which can be performed using an SPR system from XanTec. Note that not every SPR system can do these – we are quite proud of our flexible, robust and sensitive technology.

Part I: Traditional SPR and XanTec

The traditional, standard use of SPR is in the field of biological interaction analysis, otherwise known as BIA. The design of an SPR system gives them a high level of sensitivity to changes at the chip surface (see our tech notes for more detail). This means that we can track the interaction of molecules at the chip surface in fine detail, following numerous aspects of the process.

Kinetics

One of the most common SPR applications is in determining binding kinetics. In this case a molecule of interest, say a protein, is immobilised to the sensor chip. A solution containing a potential binding partner, perhaps the small molecular entity we are developing into a drug, is washed over the chip. We will see changes in resonance as the molecules interact, which provides information about the binding kinetics – switching the flow to buffer alone will then show the dissociation kinetics. Easy!

Thermodynamics

Thermodynamic calculations can be performed by leveraging the ability of XanTec SPR systems to operate at a wide range of temperatures, (up to 95°C with the correct module). Measurements of affinity constants at multiple temperatures can be used to calculate, via Van't Hoff thermodynamic analysis, changes in entropy and enthalpy during protein-protein interaction. Sound complex? Not really, it's an easy calculation to make using the data provided by XanTec's software.

Concentration

Attempting to find the concentration of an analyte, for example in the media from your latest expression run? SPR allows simpler, faster techniques than those required for a typical ELISA. The secret lies in assaying solutions under mass-transport limited conditions (in which the analyte diffusing to the chip surface is the rate limiting step). In this situation, binding rate is directly proportional to analyte concentration, and so can be easily calculated. Best of all, it is a label-free method, saving you time and money.

Fragment screening

Development of novel pharmaceuticals has long involved high throughput screening, in which thousands of small molecules are tested for their ability to inhibit a particular enzyme, process, etc. Sadly this method is not overly effective, and so many are focusing on fragment screening. In this technique thousands of even smaller molecules are tested for binding affinity, then linked together to make a synergistic, strong inhibitor. As SPR is a highly sensitive test of binding affinity, it has become commonly used in fragment screening. Simply immobilise your enzyme of interest to the chip, use lab automation to load and run your samples, and then examine the data for interesting compounds.

Determining selectivity

Epitope Mapping of antibodies
Epitope mapping involves testing the ability of pairs of antibodies to bind to a target molecule – when both recognise to a similar epitope, interference will prevent simultaneous binding. This is commonly performed by ELISA, however this can be difficult to interpret and requires labelling of one antibody. A simpler method utilises SPR, immobilising one antibody to the chip, binding the target protein to it, and then washing the second antibody across. The SPR response curve shows binding (or lack thereof) unambiguously, while the ability to use unlabelled antibodies simplifies the experiment tremendously.

Selecting binding pairs for ELISA application
How does epitope mapping help further experiments? Suppose your group wants to develop an ELISA test, which requires both an immobilisation antibody and a detection antibody. Naturally, both of these antibodies need to bind different epitopes. Fortunately, it is easy to determine non-overlapping binding pairs using the data obtained from your SPR epitope mapping.

Determining specificity

Screening for inhibitor specificity
Your group has spent months designing a diabody to target tumour cell antigens, but you need to know if specifically targets the antigen. Difficult? Simple! Immobilise your diabody to the chip, then pass a mixture of human proteins over, with and without added antigen. XanTec SPR systems then allow you to quantify just how much off-target binding is occurring, and this can then be minimised with further development.

Screening for cross-reactivity
The propensity of antibodies to bind to epitopes other than those they were raised against is known as cross-reactivity. This can be either a bonus, (e.g. vaccines against smallpox/cowpox), or a curse (when your new therapeutic antibody fails the tissue cross-reactivity test prior to Phase I). As such, determining cross-reactivity is an important part of the development process. SPR can provide many benefits in this process, ranging from mapping epitopes to quantifying the kinetics and binding specificity of these cross-reactive interactions.

Checking activity after purification
Your company has decided to improve downstream processing systems, and so are testing a number of new purification methods at lab scale. How do you verify, quantitatively, that the new system produces substrate-binding enzymes? By immobilising the substrate to a chip, the product from each purification method can be tested for binding affinity without the need for slow labelling reactions.

Part II: XanTec – Above and beyond

Quartz/Microscope flow cells

An interesting development has involved the combination of SPR flow cells which are partially made from transparent quartz, thus allowing the combination of SPR and fluorescence spectroscopy. This allows for some clever applications, such as correlating the movement of macrophages (visualised by phase-contrast), with interior alterations in organelle positioning (fluorophore expression) and the deposition of protein onto the extracellular matrix/chip surface (SPR). These quartz flow cells, available from XanTec, provide molecular biologists with powerful new quantitative tools.

Photo-reactivity

Another modular option, UV-Visible flow cells allow researchers to determine the effects of light on their processes. Take an example laboratory group, who are attempting to create biocompatible gels which can expand or contract in response to light – a first step on the road to biomimetic muscles. They need to gain detailed kinetic information on the process and so add the UV flow cell to their system, this allows real-time measurement of expansion and contraction. A wide range of photo-sensitive reactions can be followed in detail using SPR, just ask us for help.

Organic solid-phase reactions

Solid-phase reactions involve the immobilisation of one reactant to a support, often a polymer bead, and then forming further step-wise reactions on this immobilised molecule. The process has become very popular due to the ease of purifying the final product, and is the most common method used in peptide synthesis. As previously mentioned, XanTec systems work well in organic solvents and strong acid/base solutions, both of which are common conditions used in solid-phase synthesis. This means that solid-phase reactions can be performed directly on the SPR chip – for example, one can observe the progress of peptide synthesis on the chip in real time. This in turn leads to quicker characterisation and development of the solid-phase reaction process.

Metal-organic frameworks

Metal-organic frameworks (MOFs) are, simply enough, metal ions co-ordinated to organic molecules; with potential uses in hydrogen storage and catalysis. Successful development requires characterisation of binding, catalysis, and diffusion characteristics, all strengths of SPR systems. The systems sold by XanTec are well suited for use in this field as they allow the preparation of MOFs directly on the chip, ready for further characterisation.

Coupling techniques (HPLC, mass spectrometry etc)

Your tumour-fighting antibody is bound to the chip, and unfortunately there is off-target binding occurring. But what exactly is it? Luckily XanTec SPR systems, modular and designed to work with other systems, let you plug the output directly into a HPLC or mass-spectrometry system. Use the combined power of these systems to characterise and identify your unwanted binding partner simultaneously, saving time and money.