Biolayer Interferometry (BLI) is an optical biosensing technology used to analyze interactions between biomolecules. This technique provides a real-time view of how molecules bind and unbind, offering insights into dynamic biological processes. BLI provides information about the strength and speed of molecular interactions, making it a valuable tool in fields like drug discovery and fundamental biological research.
The Operating Principle
The foundation of BLI lies in the measurement of changes in the optical thickness of a biolayer on a biosensor surface. A biosensor, typically a fiber optic tip, has an immobilized molecule, known as the ligand, on its surface. When this biosensor is dipped into a solution containing another molecule, called the analyte, binding occurs if there is an interaction.
White light, which contains a broad range of wavelengths, is directed down the fiber optic biosensor. This light reflects from two surfaces: the outer surface of the biosensor tip where the biomolecules bind, and an internal reference layer within the biosensor. These two reflected light waves interfere with each other, creating a unique interference pattern.
As the analyte molecules bind to the immobilized ligand on the biosensor tip, the thickness of the biological layer increases. This increase in thickness alters the optical path length of the light reflected from the outer surface. Consequently, a shift occurs in the interference pattern’s wavelength.
A detector, such as a spectrometer, captures this shifting interference pattern in real-time. The magnitude of this wavelength shift directly correlates with the number of molecules bound to the biosensor surface. By continuously monitoring this shift, scientists can track the association (binding) and dissociation (unbinding) of molecules in real-time.
Diverse Applications
One primary application is kinetic analysis, where BLI measures the speed at which molecules bind together (association rate, or kon) and separate (dissociation rate, or koff). These rates provide insight into the dynamic nature of interactions, for example, how quickly an antibody binds to its target antigen or how long a drug candidate stays attached to a protein. This kinetic data is then used to calculate the overall binding strength, known as affinity (KD).
BLI is also commonly used for concentration determination, allowing researchers to quantify specific molecules in a sample. For instance, it can determine the amount of a particular protein or antibody present in a solution. This is achieved by comparing the binding response of an unknown sample to a standard curve generated from known concentrations.
The technology is valuable in screening applications, where researchers aim to identify potential drug candidates or binding partners from large collections of molecules. BLI can rapidly assess whether a molecule from a library interacts with a target protein, making it efficient for early-stage drug discovery. This includes fragment screening, which identifies small molecules that bind to a target.
Another specialized application is epitope binning, which characterizes how different antibodies bind to distinct regions, or epitopes, on an antigen. This helps in understanding antibody diversity and functionality, especially when developing therapeutic antibodies. BLI can also analyze interactions involving diverse biomolecules such as protein-protein, protein-nucleic acid, protein-lipid, and protein-small molecule interactions.
Key Advantages
BLI offers several advantages in molecular interaction studies. A significant benefit is its label-free detection, meaning there is no need to attach fluorescent tags or other labels to the molecules being studied. This simplifies sample preparation, avoids potential interference from the labels themselves, and allows for the analysis of molecules in their unmodified state.
Another advantage is the ability to collect real-time data, as BLI monitors binding events as they occur. This provides dynamic information about association and dissociation phases, yielding kinetic parameters like association and dissociation rates. This live monitoring offers a more complete understanding of molecular behavior compared to endpoint measurements.
BLI systems are compatible with crude samples, meaning that samples do not always require extensive purification. This can save time and resources in research, as the presence of other components in the sample often does not significantly interfere with the signal. This compatibility extends to a wide range of sample types, including proteins, nucleic acids, and viruses.
The high-throughput capability of BLI systems allows for the simultaneous analysis of many samples. Instruments like the Octet RED384 can monitor binding on up to 16 biosensors concurrently, accelerating research workflows, particularly in drug screening efforts. BLI is also user-friendly, with a relatively straightforward experimental setup and operation.