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Mass transport and interaction rates

Mass transport

Because interaction occurs at the surface of the sensor chip, analyte must be transported laterally in the flow cell from the bulk solution to the surface before interaction with ligand can take place. In a controlled flow system such as Biacore instrument, this transport is a diffusion-limited process, referred to as mass transport, that can be described by a well-defined mathematical model. If there is no mixing or replenishment of analyte (as in a well in a microplate), binding to the surface will deplete analyte from the solution close to the surface, as is illustrated in the figure below.

The situation is different in Biacore because sample is constantly replenished by constant flow. Higher flow rates replenish the analyte close to the surface more efficiently.

Interaction rates

The observed rates of interaction between analyte and ligand are the result of two processes, namely mass transport of analyte to the surface and interaction between the analyte and ligand molecules.

In a situation where mass transport and interaction rates are of similar orders of magnitude, the following considerations hold:

• Interaction processes will dominate more over mass transport in the observed binding rates at lower concentrations of available ligand, since the net rate of analyte binding to the ligand will be slower. This is the reason for recommending the lowest feasible immobilization level for compound characterization assays.
• Interaction processes will dominate more at higher flow rates, since mass transport is faster. The mass transport rate is a function of the cube root of the flow rate for diffusion-controlled transport in laminar flow systems: in other words, increasing the flow rate by a factor of 10 will approximately double the mass transport rate. Analysis of the same sample concentrations at different flow rates can help to resolve mass transport from interaction rates: the flow rate can be varied on a sample-by-sample basis in the assay setup.

A common fallacy is that the relative importance of mass transport and interaction must vary with analyte concentration. This is not true: both rates are directly proportional to analyte concentration, so the relative importance of the two processes is unaffected.

Mass transport limitations

Mass transport limitations arise when interaction at the surface consumes analyte faster than it can be supplied by mass transport or, conversely, analyte does not diffuse fast enough from the surface during dissociation, leading to re-binding.

Under the flow conditions in Biacore, the rate of mass transport of analyte to the surface varies with the cube root of the flow rate. The rate of analyte binding to ligand, on the other hand, is independent of the flow rate. Comparison of the observed binding rates at different flow rates can thus reveal whether the binding is limited by mass transport processes.

Dealing with mass transport limitation

If mass transport is the limiting factor in the rate at which analyte can bind to the surface, the measured kinetic constants will reflect the transport process rather than the interaction characteristics. To minimize the effect of mass transport, analyses should be run at a high flow rate (30 µl/minute or higher) and with a low ligand level (so absolute rate of analyte binding is low and is less likely to be limited by mass transport). Even though mass transport limitation is included in the fitting model it is important to design your experiment in such a way that mass transport limitation is minimized.

Control experiment

Perform analyses using a single analyte concentration in the middle of the concentration series over a wide range of flow rates, e.g. 5, 15, 75 µl/minute. Prepare an overlay plot of the three binding curves.

If the slope of the binding curves during sample injection differs significantly, with the slowest binding at the lowest flow rate, mass transport limitations are significant.

If the three curves show the same slope during sample injection, mass transport limitations are negligible.

If binding is very fast or very slow, it may be difficult to decide whether the slopes are significantly different. Repeat the experiment with a more suitable analyte concentration.

If the control experiment indicates that mass transport is limiting, include a term for mass transport in the model used to evaluate the data. Usually, the observed binding includes components of both transport and interaction, and the rate constants for the different processes can often be resolved. In cases where the interaction mechanism itself is complex, it may not be feasible to introduce an additional term for mass transport in the model. In such cases, it is important to reduce mass transport limitations as far as possible.

See also

List of help files

Curve fitting principles

Basic theory of kinetics