Bio-Layer Interferometry (BLI) vs Surface Plasmon Resonance (SPR) vs Grating-Coupled Interferometry (GCI) comparison

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

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

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

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.