Understanding how molecules bind and interact is fundamental in fields such as drug discovery, diagnostics, and basic research. Label-free technologies are powerful tools for studying these interactions in real-time, without the need for fluorescent tags or radioactive labels that can interfere with natural binding events. Two prominent label-free techniques widely employed for this purpose are Biolayer Interferometry (BLI) and Surface Plasmon Resonance (SPR).
How Biolayer Interferometry Works
Biolayer Interferometry measures molecular interactions by detecting changes in the optical thickness of a biosensor tip. White light reflects off two surfaces of the biosensor: an internal reference layer and an immobilized layer where molecules bind. As molecules bind to or dissociate from the biosensor surface, the optical thickness of the immobilized layer changes, leading to a shift in the interference pattern of the reflected light. This shift is directly proportional to the mass of molecules bound to the biosensor, allowing real-time monitoring of association and dissociation events.
BLI operates in a “dip-and-read” format, where biosensor tips are dipped into samples contained within microplates. This setup allows for parallel processing of multiple samples simultaneously, as each biosensor tip functions independently. The technique provides data on binding kinetics, including association (kon) and dissociation (koff) rates, equilibrium dissociation constants (KD), and concentration measurements. Its design makes it robust to complex sample matrices, as measurements are taken relative to a reference layer on the same tip.
How Surface Plasmon Resonance Works
Surface Plasmon Resonance detects molecular interactions by monitoring changes in the refractive index at a sensor surface. This technique utilizes a thin gold film coated on a glass prism, where an evanescent wave is generated when polarized light reflects off the surface at a specific angle. This evanescent wave excites surface plasmons—electron oscillations—in the gold film, causing a drop in the intensity of the reflected light at a characteristic resonance angle. Molecular binding to the sensor surface alters the local refractive index, which in turn shifts this resonance angle.
SPR systems typically employ a microfluidic flow cell, where samples are flowed continuously over the sensor chip. This precise control over sample delivery ensures consistent interaction conditions and rapid response times. SPR provides real-time data on binding kinetics, affinity, and concentration. The change in the resonance angle, measured in response units (RU), directly correlates with the mass of molecules binding to the surface.
Comparing BLI and SPR
The fundamental difference in detection lies in their physical principles; BLI measures changes in optical thickness via light interference, while SPR detects changes in refractive index at a metal-dielectric interface. BLI’s “dip-and-read” format simplifies fluidics and reduces the impact of sample matrix effects. It can accommodate crude samples like cell lysates or serum with less pre-treatment, as measurements are less sensitive to bulk refractive index changes in the solution.
In contrast, SPR relies on a continuous flow system where samples are precisely delivered over a sensor chip. This microfluidic control offers high resolution and rapid analyte exchange, but it also makes SPR more sensitive to bulk refractive index differences between samples and running buffer. Consequently, SPR often requires more extensive sample preparation and buffer matching to achieve optimal data quality.
For throughput, BLI generally excels due to its ability to process 8, 16, or even 96 biosensor tips in parallel, enabling high-throughput screening campaigns. SPR systems, while typically offering lower parallel processing capabilities, are recognized for their high sensitivity and the quality of their kinetic data, particularly for very low molecular weight analytes or weak interactions. The continuous flow provides precise control over binding events, leading to high-resolution kinetic plots.
Maintenance and operational complexity also differ; BLI systems are generally simpler to operate and maintain, with fewer issues related to clogging or microfluidic integrity compared to SPR systems.
Selecting the Right Technique
Choosing between BLI and SPR depends on specific experimental goals, sample characteristics, and available resources. BLI is often preferred for high-throughput screening, such as in early drug discovery phases where many compounds need to be screened. Its robustness to crude or complex samples also makes it suitable for applications involving cell lysates or unpurified proteins, reducing the need for extensive sample preparation. BLI can also be a more accessible solution for quick answers or when budget is a concern.
Conversely, SPR is frequently selected when high-resolution kinetic data and precise affinity measurements are paramount. Its precise fluidics are advantageous for detailed mechanistic studies of binding, including complex binding models or very fast kinetic events. If working with very low sample volumes or requiring high sensitivity for small analytes, SPR may provide the necessary resolution. The optimal choice aligns with the specific scientific question, sample nature, and laboratory capabilities.