Surface plasmon resonance (SPR)

SPR has seen considerable growth in popularity since its commercialization in the 1990s,^18–20 providing label-free quantification of protein–ligand affinities and kinetics.^1,21 In an SPR experiment, the analyte (or ligand) is immobilized to a sensor surface—usually a thin gold layer on a glass support (Fig. 2a). The sensor surface is then illuminated with polarized light at an angle that excites surface plasmons, known as the SPR angle. Small changes in the refractive index of the sensor surface (e.g. caused by molecules binding to the surface) will affect the SPR angle and the detected light intensity, which is reported in response units (RU). Therefore, RUs are proportional to the amount of ligand bound to the analyte on the sensor surface.

After the immobilization step, the analyte is exposed to a continuous flow of ligand. Ligand molecules binding to the analyte will change the SPR angle and lead to an increase in RU, yielding an association curve (Fig. 2b). Once the RUs are constant, either all binding sites on the surface are saturated or the system has reached chemical equilibrium. At this point, the ligand solution is replaced by blank buffer, which makes dissociation the dominating process. This results in a decrease in RUs, yielding a dissociation curve. Both association and dissociation curves are characterized by the on- and off-rates of the interaction. Knowing the concentration of molecules in solution and fitting the binding curves, based on the Langmuir isotherm, provides k_on and k_off and consequently also the K_d. Although different binding models are available, recent literature mostly relies on 1 : 1 interaction models, implying that SPR is mostly used to characterize simple stoichiometries.

The strengths of SPR are its large dynamic range for K_d measurements (sub-nM to low mM) and the small sample amounts required (several μg per sensor chip).⁶ Experimental conditions are compatible with different buffers, although care needs to be taken when using detergents, chelating agents or denaturing agents. Measurements are fully automated and the sample preparation steps needed are minimal.

Despite the high degree of automation, SPR remains a mostly low-throughput method because several runs are needed to obtain robust measurement of kinetics and binding affinities, which can take several hours. Further limiting factors are the requirement that the ligand be immobilized to the sensor surface;⁶ mass transport effects, which limit the upper limit of accessible kinetic processes (k_off < 10⁻¹ s⁻¹); and sensitivity of SPR to non-specific interactions between sensor surface and analyte.²⁵

Despite these limitations, SPR is extremely popular due to being user-friendly and having broad applicability to various biomolecule classes. Given the large number of SPR publications, we refer here to reviews showcasing recent developments in SPR applications for protein–protein interaction quantification,^26–28 high-throughput with SPR imaging sensors (SPRi),²⁹ sensitivity and detection speed,^30,31 influence of capture surfaces,³² and overcoming challenges with multi-valent binding.^33,34

Mamer et al., in their review, show SPR's convergence to cell-based protein–protein interaction measurements.³⁵ Recent research focusing on SARS-CoV-2 protein interactions highlights the applicability of SPR, as shown in Fig. 2c, where Wrapp et al. quantified the affinity of the SARS-CoV-2 spike protein (violet) binding to neck-domain-free ACE2 (blue).²² His-tagged S protein was immobilized to a Ni–NTA sensorchip and exposed to serial dilutions of untagged ACE2 (250 to 15.6 nM), each yielding a sensorgram (black lines). The experimental data was fitted with a 1 : 1 Langmuir model (red), revealing binding kinetics of k_off = 2.76 × 10⁻³ s⁻¹ and k_on = 1.88 × 10⁵ M⁻¹ s⁻¹, and a K_d of 14.7 nM. Future work is directed towards increasing SPR's sensitivity and specificity—which will further expand its applicability to various classes of biomolecules and their interactions.