Biolayer Interferometry (BLI) is an optical technology used to study biological molecular interactions in real-time. This label-free technique monitors how one molecule binds to another without fluorescent tags or radioactive labels. By measuring changes in light interference, BLI delivers quantitative data characterizing the binding strength and speed of molecular complexes. This method determines binding affinity (complex stability) and binding kinetics (rates of association and dissociation).
The Principle of Interference
The core of Biolayer Interferometry lies in the physics of thin-film interference, a phenomenon where light waves interact after reflecting off two closely spaced surfaces. In a BLI system, a beam of white light is directed down a fiber optic biosensor tip. This light encounters two reflective surfaces: a fixed internal reference layer and an outer layer where the target molecule, called the ligand, is immobilized.
When light reflects from these two surfaces, the resulting waves travel slightly different distances, causing them to recombine and interfere. This interference creates a characteristic spectral pattern recorded by the instrument. This pattern remains stable unless the optical thickness between the two reflective surfaces changes.
Molecular interaction measurement occurs when the biosensor tip is dipped into a solution containing the second molecule, known as the analyte. As the analyte binds to the immobilized ligand, it adds mass and increases the optical thickness of the biolayer. This thickness change alters the light path, causing a measurable shift in the reflected light’s interference pattern, registered as a change in wavelength.
The magnitude of this wavelength shift is directly proportional to the mass of the bound analyte molecules. This continuous, real-time monitoring of the wavelength shift is the direct readout of the binding event. As molecules bind, the wavelength shifts in one direction; when they dissociate, the wavelength shifts back.
Essential Components of a BLI System
A BLI system is fundamentally built around a set of disposable fiber optic biosensors and a precise reader instrument. The biosensors consist of a glass fiber coated with a biocompatible matrix designed to immobilize the ligand. These tips are typically functionalized with specific coatings, such as streptavidin, which allows for the stable capture of a biotinylated ligand molecule.
The system utilizes a unique “dip and read” methodology, which contrasts with other label-free techniques that rely on complex internal fluidic systems. Biosensor tips are robotically dipped sequentially into microplate wells containing the necessary buffers and sample solutions. This physical movement eliminates the need for microfluidics, which are often prone to clogging and require extensive cleaning.
The reader instrument houses the optical components, including the white light source and the spectrometer that measures the interference pattern. This instrument precisely controls the movement of the biosensor tips and records the wavelength shift data from multiple tips simultaneously. The ability to measure several interactions in parallel contributes to the technology’s high-throughput capability.
Analyzing Molecular Interactions
The primary output of a BLI experiment is the sensorgram, a graphical representation that plots the wavelength shift (in nanometers) against time. This curve is generated in two main phases: the association phase, where the sensor tip is immersed in the analyte solution, and the dissociation phase, where the tip is moved to a buffer-only solution, allowing the bound molecules to naturally unbind.
The shape of the sensorgram provides the raw data necessary to calculate the quantitative parameters of the molecular interaction. During the association phase, the curve rises as the analyte binds to the ligand, reflecting the speed of complex formation, known as the association rate constant (\(k_{on}\)). Conversely, the slope of the curve during the dissociation phase quantifies the stability of the complex, yielding the dissociation rate constant (\(k_{off}\)).
These two kinetic rate constants are then used to calculate the binding affinity, represented by the equilibrium dissociation constant (\(K_D\)). The \(K_D\) value is derived from the ratio of \(k_{off}\) to \(k_{on}\) and measures how strongly two molecules interact. A smaller \(K_D\) value signifies a higher binding affinity, indicating a more stable molecular complex.
Beyond characterizing binding kinetics and affinity, BLI is also used for the determination of molecular concentration. The instrument measures the total mass of the analyte bound to the sensor tip within a fixed period, generating a signal directly proportional to the analyte’s concentration. By referencing a standard curve of known concentrations, the system can rapidly quantify the amount of a target molecule in a complex solution.
Operational Advantages for Drug Discovery
The design of the BLI system offers several practical benefits that make it highly suitable for drug discovery and development. The core advantage is its capacity for high throughput screening, achieved through the ability to run multiple sensor tips simultaneously across a 96-well or 384-well microplate format. This parallel processing greatly accelerates the screening of compound libraries or antibody panels, shortening the timeline for identifying promising drug candidates.
The “dip and read” format contributes significantly to operational simplicity and robustness. Since there are no complex microfluidic channels, the system is highly tolerant of unpurified or “crude” samples, like cell culture supernatants or cell lysates. Researchers can analyze samples with minimal preparation, which is a substantial time-saver.
The absence of a fluidic system also means the measurements are less sensitive to variations in the sample matrix, such as changes in viscosity or refractive index. This inherent stability ensures that the measured signal is a true reflection of the molecular binding event, rather than an artifact of the sample’s physical properties.