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SPR, ITC, MST & BLI: What’s the optimal interaction technique for your research?

Binding data is key for publications. There are many techniques available to quantify biomolecular interactions that provide the binding affinity, kinetics and/or thermodynamics. This data is important for publications because it allows for an in depth characterization of biological interactions. Binding affinity describes how strong the interaction is between two biomolecules. Although the binding affinity is an important number, it doesn’t tell the whole story. As we mentioned in a previous blog post, affinity is really only the tip of the iceberg when it comes to understanding the nature of an interaction. Two other very important aspects to examine are kinetics (how fast the interaction happens) and specificity (how specific the interaction is between the two molecules). This type of information is critical for understanding the biological system that is being studied, especially for applications like drug discovery and understanding molecular disease mechanisms.

Which biomolecular interaction technique best suits my research?

Currently, the four most used techniques are Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC), Microscale Thermophoresis (MST), and Biolayer Interferometry (BLI). Each technique has its advantages and disadvantages. Read the SPR, ITC, MST & BLI comparison below and take this 5 minute quiz to determine the optimal technique for your research:

Surface Plasmon Resonance (SPR)

Surface plasmon resonance (SPR) is a label-free technology that allows researchers to quantitatively analyze binding between two biomolecules. SPR typically consists of sensors made out of thin films of gold which are key for this label-free technique. Incident light is used to collectively excite electrons of a conduction band in a metal, creating a coherent plasmon oscillation. The plasmon resonance changes with binding events to the gold film due to a change in the refractive index, which is measured in real-time and results in a binding curve. Various concentrations of analyte are used to get a few binding curves which are fit to a binding model to determine kinetic binding constants such as the kon, koff and KD.

Advantages:

  • Label-free detection
  • Real-time data (i.e. quantitative binding affinities, kinetics and thermodynamics)
  • Medium throughput
  • Sensitivity and accuracy
  • Measures over a very wide range of on rates, off rates and affinities
  • Small sample quantity

Disadvantages:

  • Expensive instrument and sensors
  • Expensive maintenance
  • Steep learning curve
  • Specialized technician or senior researcher required to run experiments
  • Immobilization of one of the binding partners required

Localized Surface Plasmon Resonance (LSPR)

Localized surface plasmon resonance (LSPR) has been studied for many years and is an accessible and affordable alternative to SPR, which is why Nicoya Lifesciences bases their OpenSPR instrument on LSPR. LSPR is generated by metal nanoparticles, typically gold and silver, instead of a continuous film of gold which is used in traditional SPR. LSPR produces a strong resonance absorbance peak in the visible range of light, with its position being highly sensitive to the local refractive index surrounding the particle. Therefore, LSPR measures small changes in the wavelength of the absorbance position, rather than the angle as in traditional SPR.

Advantages:

Using gold nanoparticles instead of a thin film of gold allows LSPR to have the same advantages as SPR and many more including:

  • Affordable instrument and sensor chips  
  • Portable
  • Easy maintenance
  • Robust against vibration and noise
  • Less interference from buffer mismatch and temperature drift

Disadvantages:

  • Immobilization of one of the binding partners required
  • Lower throughput compared to complex, highest throughput level instrument models

Isothermal Titration Calorimetry (ITC)

Isothermal Titration Calorimetry (ITC) is a technique used for quantitative thermodynamic characterization of a wide variety of biomolecular interactions. It works by directly measuring the binding equilibrium by determining the heat evolved on association of a ligand with its binding partner.

Advantages:

  • Ability to determine thermodynamic binding parameters (i.e. stoichiometry, association constant, and binding enthalpy) in a single experiment
  • Modification of binding partners are not required

Disadvantages:

  • Large sample quantity needed
  • Kinetics (i.e. association and dissociation rate constants) cannot be determined
  • Limited range for consistently measured binding affinities
  • Non-covalent complexes may exhibit rather small binding enthalpies since signal is proportional to the binding enthalpy
  • Slow with a low throughput (0.25 – 2 h/assay), not suitable for HTS

Microscale Thermophoresis (MST)

Microscale Thermophoresis (MST) is a technique that measures the motion of molecules along microscopic temperature gradients and detects changes in their molecule size, charge, and hydration shell.

Advantages:

  • Small sample size
  • Immobilization free
  • Minimal contamination of the sample (method is entirely optical and contact-free)
  • Ability to measure complex mixtures (i.e. cell lysates, serum, detergents, liposomes)
  • Wide size range for interactants (ions to MDa complexes)

Disadvantages:

  • Hydrophobic fluorescent labelling required, may cause non-specific binding
  • No kinetic information (i.e. association and dissociation rates)
  • Highly sensitive to any change in molecular properties

Biolayer Interferometry (BLI)

Bio-Layer Interferometry (BLI) is an optical analytical technique used to quantify biomolecules which are typically adsorbed to the tips of optical fibers by analyzing the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip and an internal reference layer.

Advantages:

  • Label-free detection
  • Real-time data
  • No reference channel required
  • Crude sample compatibility
  • Fluidic-free system so less maintenance needed

Disadvantages:

  • Immobilization of ligand to surface of tip required
  • No temperature control
  • Low sensitivity (100-fold lower sensitivity of detection compared to SPR)

Conclusion

Now that you’ve learned about the advantages and disadvantages of SPR, LSPR, ITC, MST, and BLI, you can decide which interaction technique is best fit for your research. After comparing the four most commonly used techniques for quantifying biomolecular interactions, each has its advantages and disadvantages but SPR proves to be the most versatile technique. However, most researchers don’t have access to this technology due to the complexity and cost of the instrument. Fortunately, Nicoya Lifesciences’ OpenSPR uses LSPR technology to make the technique more accessible and affordable than traditional SPR. OpenSPR provides researchers with these main benefits:

1. Benchtop

Avoid costly & inconvenient core facilities with our affordable benchtop solution

2. Real-Time Data

Publish faster with label-free binding kinetics & affinity data

3. User-Friendly

Train anyone in your lab to become an SPR expert with our user-friendly solution

4. Low Maintenance

Forget about expensive service contracts so you can focus on your research

Find out how benchtop SPR can help you publish sooner:

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