Binding constant calculation using surface plasmon resonance (SPR) and measures against nonspecific adsorption

Understanding Surface Plasmon Resonance (SPR)

Surface Plasmon Resonance (SPR) is a powerful and versatile technique used to measure the interaction between molecules in real-time without the need for labeling.
This optical method is based on the detection of changes in the refractive index near a sensor surface, which occurs when molecules bind to the surface.
SPR is highly sensitive and can provide valuable information on binding kinetics, affinity, and concentration of analytes.

How SPR Works

In an SPR experiment, a thin metal film, often gold, is coated on a glass slide and serves as the sensor surface.
When polarized light is shone onto the sensor at a specific angle, surface plasmons, which are electron charge density waves, are excited at the metal-dielectric interface.
This causes a drop in the reflected light intensity, known as the SPR dip.
When molecules bind to the sensor surface, the refractive index changes, causing a shift in the SPR angle or resonance condition.
The shift is detected in real-time and is proportional to the mass of the bound molecules.

Calculating Binding Constants with SPR

SPR is particularly advantageous for calculating binding constants as it provides real-time association and dissociation data.
To determine these constants, an experiment is designed wherein one interaction partner (ligand) is immobilized on the sensor surface while the other (analyte) is flowed over it.

Association and Dissociation Phases

The association phase begins when the analyte is introduced and starts to bind to the ligand on the sensor surface.
As binding progresses, a sensorgram, which plots response units (RU) against time, is generated.
The slope of the sensorgram during association can be analyzed to determine the association rate constant (ka).
The dissociation phase occurs when the analyte is flushed away and no longer renews its binding to the ligand.
This phase is analyzed to obtain the dissociation rate constant (kd).

Equilibrium and Affinity Constants

The equilibrium dissociation constant (KD), an important parameter representing the affinity of the interaction, is calculated using the formula KD = kd/ka.
A lower KD value indicates a higher binding affinity, suggesting that the interaction is strong and stable.

Challenges in SPR: Nonspecific Adsorption

While SPR is a robust technique, nonspecific adsorption can pose significant challenges.
This occurs when the analyte binds to unintended sites on the sensor surface, creating background noise and skewing results.

Identifying Nonspecific Adsorption

Nonspecific adsorption is identified by noting responses that deviate from expected interaction profiles or through unexpected changes in the sensorgram’s baseline.
It can lead to inaccurate measurements of binding kinetics and affinity.

Measures Against Nonspecific Adsorption

To combat nonspecific adsorption, several strategies can be employed:

1. **Surface Blocking**: The sensor surface can be treated with blocking agents like Bovine Serum Albumin (BSA) to minimize unscheduled interactions.

2. **Optimizing Buffer Conditions**: Carefully optimizing the composition of the running buffer can help reduce nonspecific binding.
Buffers often contain surfactants like Tween 20 to deter unwanted adsorption.

3. **Proper Sensor Surface Preparation**: Ensuring the ligand is immobilized uniformly and that the surface chemistry is compatible with the analyte can help diminish nonspecific binding.

4. **Increasing Stringency**: Adjusting the ionic strength or pH of the buffer can sometimes resolve issues with nonspecific binding by destabilizing weak, undesirable interactions.

Conclusion

Surface Plasmon Resonance (SPR) is a crucial tool in the study of molecular interactions, particularly in the calculation of binding constants.
Despite the challenge of nonspecific adsorption, careful experimental setups, buffer optimizations, and surface preparations can mitigate its effects, allowing researchers to gain precise and valuable insights into biomolecular binding events.
Overall, SPR remains a cornerstone technique in biochemistry and molecular biology, providing a window into understanding interactions at the molecular level.