Bio-layer Interferometry vs Surface Plasmon Resonance vs Grated Coupled Interferometry

Which biomolecular interaction techniques are used in research?

Understanding the interaction between molecules – particularly the kinetics – can answer many questions, so it’s no surprise that it needs to be analyzed in many areas of research.

For instance, you may need to know how a molecule interacts with a receptor to understand how signaling occurs in an organism. Or you may want to understand whether, and how tightly, a drug binds to a compound of interest for drug discovery. The binding affinity can tell us this, but we can get more detail from measuring the binding kinetics.

The applications where binding kinetics are relevant, are as broad as the technologies, and several technologies can be used to study them. Here we compare three.

Note: for more information about measuring interactions, thermodynamics and more, please see our ITC technology page.

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What is Biolayer Interferometry?

Biolayer Interferometry (BLI) is an optical, surface-based, label-free technology. Unlike other biosensor technologies, BLI does not work with a microfluidic flow, but by immersion of sensor tips into the sample/buffer. Light reflected off the tip of an optical fiber exhibits a phase shift depending on the refractive index near the tip surface. The reflected light interferes with light reflected off an internal reference surface.

By using white light as a source, a spectral interference pattern is recorded, which comprises information about the refractive index near the tip surface. As biomolecules bind to the biolayer surface immersed in the experimental solution (such as a sample), the refractive index profile and thus the spectral pattern changes.

Advantages

Easy to use
Virtually clog-free for complex and sticky samples
Bulk effects minimized using reference tips

Disadvantages

Sensors are orders of magnitude less sensitive than SPR and GCI sensors
Limited accuracy when determining kinetic parameters
Limited ability to measure tight binders and fast on-rates; measurements limited by diffusion
Limited ability to measure fast off-rates

What is Surface Plasmon Resonance?

Surface plasmon resonance (SPR) is another optical, label-free analysis method – in fact, it was one of the first surface-based label-free technologies. SPR detects refractive index changes caused by molecular interactions within an evanescent field near a sensor surface.

In these sensors, a metal film on a glass support is illuminated with light of a specific wavelength. At a specific angle, depending on the refractive index close to the surface, so-called surface plasmons are excited. Since that energy is missing in the reflected light beam, a ‘dip’ in intensity is formed when projecting onto the sensor.

By determining the position of the dip in real-time, SPR measures changes in the refractive index on the metal surface. The solutions containing the analyte are injected using microchannels, and at least one reference flow cell is used for eliminating bulk effects.

Advantages

Reference flow cells eliminate bulk effects
Can measure tight binders and fast on-rates

Disadvantages

Limited detection of fastest transitions due to use of flow cells in series
Traditional microfluidics requires high maintenance due to clogging
Limited ability to measure fast off-rates

What is Grating-Coupled Interferometry (GCI)?

Based on waveguide interferometry – another optical label-free method – Grating-Coupled Interferometry (GCI) can monitor and characterize molecular interactions in real-time, determining kinetic rate parameters, affinity constants, and concentrations of analyte molecules interacting with an immobilized ligand.

In waveguide interferometry, changes in refractive index are measured within the evanescent field of a waveguide near a sensor surface. These changes cause the light phase to change too. The light travels throughout the waveguide, creating an evanescent wave that spans the entire length of the sensor surface. The phase changes are displayed interferometrically. Creoptix’s GCI technology takes the benefits of waveguide interferometry and eliminates its typical alignment issues:

Advantages

High primary sensitivity for label-free interaction analysis
No-clog microfluidics
Can measure tight binders and fast on-rates
Can measure kinetics with fast off-rates

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BLI vs SPR vs GCI: Which biomolecular interaction technique is the best?

The best biomolecular interaction technique will depend on the application and the user’s goals. Below, you can see how these three techniques compare for four key requirements: a broad application range, measurement of weakest binders, measurement of tightest binders, and low system maintenance.

	Grating-Coupled Interferometry (GCI)	Surface Plasmon Resonance (SPR)	Biolayer Interferometry (BLI)
Broadest application range Suitable for a variety of molecules ranging from low to high molecular weights, purified or crude.	Yes Suitable for Fragments, Small Molecules, Peptides, Proteins, Viruses, Cell Culture Supernatants, Serums, Cell lysates	No Suitable for Small Molecules, Peptides (limited suitability for Fragments, Viruses, Cell Culture Supernatants, Serums, Cell lysates)	No Suitable for Cell Culture Supernatants, Serums, Cell lysates (limited suitability for Peptides, Proteins, Viruses)
Measure weakest binders Ability to measure kinetics with fast off-rates thanks to fast fluidics and high acquisition rates.	Yes Off-rates up to k_d=10 s^-1	No Off-rates up to k_d=1 s^-1	No Off-rates up to k_d=0.1 s^-1
Measure tightest binders Ability to accurately measure kinetics even for tight binders and fast on-rates.	Yes Measurement under flow conditions	Yes Measurement under flow conditions	No Measurement under diffusion-limited conditions (no microfluidics)
Less downtime Little downtime due to service or unexpected repairs.	Yes No-clog microfluidics	No Traditional microfluidics	Yes No microfluidics