Introduction
Surface plasmon resonance instruments provide a wealth of information — kinetics, affinity, and concentration. However, the type of experimental setup directly determines what data is obtainable. There are two types of setup, each designed to perform either steady-state or kinetic measurements.
Steady-state measurements can be achieved via manual injection to obtain affinity data (KD), whereas kinetic analysis requires a pump to determine both kinetic rate constants and affinity (ka, kd, KD). The resulting sensorgrams appear differently as well. This technical note explains the differences between manual injection mode and pump-assisted mode, and when to use each.
Manual Injection — Steady-State
Sample introduced by syringe. No continuous flow. Measures binding at equilibrium across a concentration series. Provides KD only. Ideal for screening, quantification, and portability-first workflows.
Pump-Assisted — Kinetic
Continuous flow via peristaltic pump. Resolves full association and dissociation phases. Provides ka, kd, and KD. Required when drug residence time or mechanistic binding data is needed.
Basic Principles of Kinetics and Affinity
In an SPR experiment, ligands (L) are immobilized onto the sensing surface and introduced to an analyte (A). If A has affinity for L at a 1:1 ratio, one can assume a Langmuir binding model:
Here, ka (M−1 s−1) is the association rate constant and kd (s−1) is the dissociation rate constant (also known as kon and koff, respectively). The dissociation equilibrium constant KD — the point at which half of the surface-immobilized ligands are bound to analytes — is expressed as:
A sensorgram has three main regions used in both steady-state and kinetic experiments. The manual injection mode (steady-state) uses the association region (A) and steady-state plateau (B) to obtain affinity data only. The pump mode (kinetics) uses the association and dissociation phases (A and C) to obtain both kinetic and affinity data.
Manual Injection Mode — Steady-State Measurement
In manual injection mode, samples are injected into the SPR instrument via syringe. Steady-state measurements involve observing equilibrium binding — where the net rate of binding is zero — as a function of analyte concentration, to determine KD.
A typical experiment involves injecting a series of increasing analyte concentrations (at least 5 concentrations) and allowing the sample to remain in contact with the sensor surface until the binding curve levels out at steady state. There is no dissociation phase, since steady-state conditions must be met and there is no flow. The SPR response at steady state is then plotted against analyte concentration to generate a binding isotherm, and KD is determined by fitting that curve to the steady-state equation.
Pump-Assisted SPR — Kinetic Measurement
A pump is required for kinetic measurements because a continuous flow must be provided to observe the dissociation phase following sample injection. A peristaltic pump is connected to the SPR instrument to deliver sample and running buffer in sequence.
Once the sensorgrams are collected, the association phase is fitted with a suitable binding model — usually a 1:1 Langmuir model — to obtain ka. The dissociation phase is fitted with a single exponential decay to find kd. KD is then calculated as KD = kd / ka. The steady-state plateau is not observed in kinetic analysis because running buffer is introduced before equilibrium is reached, forcing dissociation.
Multi-Cycle vs. Single-Cycle Kinetics
Kinetic analysis can be performed in two ways:
Multi-cycle kinetics — one analyte concentration per injection cycle, followed by a regeneration step. Each concentration provides one complete sensorgram. At least 5–8 concentrations are recommended.
Single-cycle kinetics — multiple analyte concentrations (low to high, up to 5) injected within the same cycle with no regeneration in between. Useful when regeneration is difficult or when sample volume is limited.
Comparison Summary
| Feature | Manual Injection | Pump-Assisted |
|---|---|---|
| Measurement type | Steady-state | Kinetic |
| Data obtained | KD (affinity) | ka, kd, KD (kinetics + affinity) |
| Flow | No continuous flow | Continuous flow required |
| Dissociation phase | Not observed | Observed and fitted |
| Sample volume | Low | Higher |
| Equipment needed | Syringe only | SPR instrument + pump |
| Mass transfer effects | None | Possible at high concentrations |
| Portability | Maximum — no accessories required | Reduced — pump required |
Besides the type of data needed, factors such as cost, sample volume, time, and portability all influence which setup is most appropriate for a given experiment.
The P4SPR and P4PRO — Built for Both
Affinité Instruments designed the P4SPR to accommodate both steady-state and kinetic measurements. Syringes can be used for manual injection (steady-state mode). For kinetic analysis, the AffiPump or a compatible injection loop integrates directly to deliver samples under continuous flow.
The P4PRO extends this further with integrated fluidics — enabling fully automated multi-concentration kinetic runs with reference channel subtraction and clean dissociation phases, every time.
References
- P. Anton van der Merwe, "Surface Plasmon Resonance," in Protein-Ligand Interactions: Hydrodynamics and Calorimetry, Oxford University Press, 2001.