Tips For Users Series - #1
Welcome to a new series of Surface Plasmon Resonance Insider blog posts that are focused on providing tips to maximize the quality of your SPR data. While the actual process of using Reichert Life Sciences’ SPR products is easy, there are many “tricks of the trade” that we will share with you so that you can obtain the highest quality data possible.
Tip 1: Identify and minimize the influence of mass transport
What is mass transport, and why is it a problem?
In the context of SPR, mass transport refers to the transfer of analyte from the bulk solution to the immobilized target on the sensor chip surface. The transport of analyte to the sensor surface occurs by both convection and diffusion. However, the flow within the flow cell of an SPR system is laminar (not turbulent). This leads to parabolic-shaped flow, with the highest velocity in the center and decreased velocity along the walls, resulting in a velocity of effectively zero near the sensor chip surface. This is called the unstirred boundary level. With buffer flow, the analyte is brought close to the sensor surface but then has to diffuse through the unstirred layer to reach the immobilized target. When using a 3D dextran hydrogel sensor chip, the transport also includes the diffusion of the analyte within the dextran layer; this is not a concern when using a planar sensor chip.
When the rate of mass transport is equal to or slower than the interaction association rate constant, the binding kinetics will be mass transport limited. This can lead to inaccurate data, resulting in slower apparent association rate constants. The dissociation rate can also be affected but in this case we are referring to the transfer of analyte away from the sensor chip surface. If this transfer is extremely slow, the probability of analyte rebinding to available binding sites on the target molecule increases and the observed dissociation rate is often slower than the true rate.
How do I identify mass transport?
Mass transport-limited sensorgrams tend to have little or no curvature in the association phase. This is problematic because rate information obtained from such association curves are skewed, thus yielding association rate constants that are slower than the true rates. A good way to verify mass transport limitation is to carry out the interaction experiment at different flow rates and observe how the association rate changes. Typically, if the association rate increases with higher flow rates, the interaction is mass transort limited.
An example of mass transport limited sensorgarms is shown in the Figure below:
As seen from the data, there is little to no curvature in the association phases in any of the concentration injections.
How do I minimize the influence of mass transport?
Since mass transport is a function of convection and diffusion, one way to increase the transport of analyte is to run the assay at a higher flow rate, which increases the rate of convection transport. In fact, it is recommended that kinetic experiments be carried out using a flow rate of at least 25 µL/min. However, as mentioned above, since we have laminar flow above the sensor chip surface, there is an unstirred boundary layer that the analyte has to diffuse through. As such, the best way to minimize the effect from this phenomenon is to immobilize the target molecule at a very low density on the sensor chip surface. This will lead to an analyte response that is lower, but the association kinetics will be much more accurate. In addition, the probability of analyte rebinding during the dissociation phase is minimized at low target immobilization levels so the dissociation kinetics also will be more accurate.
There are instances where the above modifications cannot fully eliminate the mass transport effect. In these cases, a fitting model can be utilized to take into account mass transport, the most common of which is the two-compartment model. In this model, binding is treated as a two-step process: the analyte is first transported to the sensor surface, and then binding occurs (i.e., reaction kinetics). This model is generally only needed for cases of relatively rapid interactions.
We hope you enjoy this column and return regularly for future posts, which will provide additional tips for your SPR experiments. You can read more about SPR elsewhere on Reichert’s website, including our first blog post and an earlier blog post on how to choose the right sensor chip: 2D or 3D.
If you would like advice on determining how to eliminate mass transport or how to fit your data, please do not hesitate to contact us.
We also ask you to provide your own input and suggestions to make this column even better. Contact us if you have any questions or topics you would like to discuss, or if you have certain tips of your own that you would like to share.
References for More Information on this Topic
• David G. Myszka, Xiaoyi He, Micah Dembo, Thomas A. Morton, and Byron Goldstein, “Extending the Range of Rate Constants Available from BIACORE: Interpreting Mass Transport-Influenced Binding Data”, Biophysical Journal, Volume 75, 1998, pp. 583–594.
• Peter Schuck, “Kinetics of Ligand Binding to Receptor Immobilized in a Polymer Matrix, as Detected with an Evanescent Wave Biosensor. 1. A Computer Simulation of the Influence of Mass Transport”, Biophysical Journal, Volume 70, 1996, pp. 1230-1249.