Introduction
Surface plasmon resonance instruments provide a wealth of information such as kinetics, affinity, and concentration. However, one must be mindful that the type of an SPR experimental setup can affect the type of data that one can obtain. There are two types of setup which are designed to perform either steady-state or kinetic measurements. Steady-state measurements can be achieved via manual injection to obtain affinity data, whereas kinetic analysis requires a pump to determine both kinetic and affinity data (Fig. 1). Thus, the resulting sensorgrams would appear differently as well. This blog will explain the differences in manual injection mode vs. a pump-assisted mode to obtain affinity and kinetic data. Please see
for more details.
Figure 1. A. Affinity data can be obtained from steady-state measurements via manual injections. B. Kinetic and affinity data can be obtained from kinetic analysis via a pump-assisted setup (e.g. peristaltic pump). Note the difference in sensorgram shape between A and B.
Basic Principles of Kinetics and Affinity
Let us start with the general idea that ligands (L) are immobilized onto an SPR sensing surface and are introduced to an analyte (A). If A has an affinity for L at a 1:1 ratio, then one can assume a Langmuir binding model:
with ka (M-1 s-1) and kd (s-1) as rate constants (also known as kon and koff, respectively) in the forward (association) and backward (dissociation) reactions, respectively. Then, the dissociation equilibrium constant, Kᴅ, which is the point at which half of the surface-immobilized ligands are bound to analytes, can be expressed in terms of the free analyte [A], ligand [L], and analyte-ligand complex [AL] concentrations as well as in terms of the rate constants.
Below is an illustrated sensorgram that represents the 3 main regions used in both steady-state and kinetic experiments. The manual injection mode (steady-state) involves the use of the association (region A) and steady-state phases (B) to obtain only affinity data, whereas the pump mode (kinetics) uses the association and dissociation phases (A and C) to obtain kinetic and affinity data.
Fig. 2. The association (A), steady-state (B), and dissociation (C) regions of the sensorgram.
Manual Injection Mode for Steady-State Measurement
The setup in the photo below is used for steady-state measurements via manual injection with Affinité’s SPR instrument where samples are manually injected into an SPR instrument via a syringe. Steady-state measurements involve the observation of equilibrium binding (net rate of binding is zero) as a function of analyte concentrations to determine the Kᴅ value, which is a measure of the strength of binding. A typical experiment involves injecting a series of increasing analyte concentrations (at least 5 concentrations) and allowing the samples to be in contact with the sensor surface to reach steady-state (where the binding curve levels out) (Fig. 1A). Notice that there would not be a dissociation phase since steady-state conditions need to be met and there is no flow. Then, the SPR response at steady-state can be plotted against analyte concentration to obtain a calibration curve. Kᴅ can be determined by fitting that curve into the steady-state equation, which relates the SPR response to analyte concentration and Kᴅ.
Fig. 3. Photo of Affinité’s P4SPR™ with manual injection.
Pump-Assisted SPR Experiment for Kinetic and Affinity Data
Fig. 4. Photo of Affinité’s P4SPR™ connected to a peristaltic pump.
Pumps are required for kinetic measurements since a continuous flow must be provided to observe the dissociation phase with time following sample injection (association phase) (Fig. 1B). Fig. 4 shows how a pump is connected to an SPR instrument to collect kinetic data. Once the sensorgrams are collected, the association phase is fitted with a suitable binding model (usually based on a 1:1 interaction Langmuir model, single exponential) to obtain the ka value, and the dissociation phase is then fitted with the single exponential decay model to find kd. Thus,Kᴅ can then be determined from Kᴅ = kd/ka. The steady-state phase is not observed in kinetic analysis because the wash buffer is flowed before the analyte in contact with the ligand-immobilized surface reaches equilibrium, forcing the analyte to dissociate from the ligands.
Kinetic analysis can be performed in two separate ways. One is called multi-cycle kinetics and the other is called single-cycle kinetics. In multi-cycle kinetics, one analyte concentration is injected to provide one complete sensorgram, followed by a regeneration step. Then, a new analyte concentration is injected to collect another sensorgram. This is repeated until all the analyte concentrations are run. A single-cycle measurement runs multiple analyte concentrations (usually from low to high) within the same cycle with no regeneration steps in between. It is recommended that various concentrations (at least 5-8 for multi-cycle and up to 5 for single-cycle) of the analyte sample should also be used to eliminate any possibilities of artifacts due to concentration dependency when fitting into a binding model [1]. The selection of concentrations should be centered around the Kᴅ (from 0.1 to 10 x Kᴅ).
Other considerations about kinetic measurements are the requirement for more sample and they are subject to mass transfer effects.
The features of performing a manual injection vs. a pump-assisted SPR experiment are summarized in the table below.
Besides considering the type of data one wants to collect, factors such as costs, amount of sample volume, time, and portability can also determine the type of experiment that one can perform.
The P4SPR™ Mode Duality
Affinité Instruments has anticipated the need for researchers to do either steady-state and/or kinetic measurements. The portable P4SPR instrument can accommodate both kinds of measurements. Syringes can easily be used to inject samples directly into the P4SPR. Alternatively, a pump, such as a peristaltic pump or our up-and-coming KNX2™, which is a injection loop module, can be integrated into the P4SPR to deliver samples.
Thank you for reading and watch out for additional blogs discussing SPR related topics such as SPR vs ELISA, wavelength vs. angle interrogation, planar SPR vs. nanoparticle localized SPR, and multiple channel SPR.
Contact us to learn more about SPR at info@affiniteinstruments.com
References
[1]. P. Anton van der Merwe. http://www.biophysics.bioc.cam.ac.uk/wp-content/uploads/2011/02/spr1.pdf [Accessed Oct. 2020].