Biolayer Interferometry (BLI) is a technology that detects the surface reaction of the sensor by detecting the displacement change of the interference spectrum; when a beam of visible light is emitted from the spectrometer, two reflection spectra are formed at the two interfaces of the optical film layer at the end of the sensor, and an interference spectrum is formed. Any changes in the thickness and density of the film layer formed by molecular binding or dissociation can be reflected by the displacement value of the interference spectrum, and a real-time reaction monitoring spectrum can be made through this displacement value.

BLI technology can detect various samples such as proteins, nucleic acids, polysaccharides, lipids, small molecule drugs, antibodies, viruses, bacteria and cells. It has been widely used in life science research fields such as protein structure target analysis, drug development and screening, immunology, gene regulation, signal pathways, genetics, microbiome, virology, nanoparticles, liposomes, Chinese medicine extracts and natural product analysis, and has won wide recognition from users.

I. Principle of BLI Technology

When light propagates in a homogeneous light source, if the propagation distance is inconsistent, an optical path difference will be generated (a). The optical path difference causes light interference, and the interference of light of different wavelengths forms an interference spectrum (b). The binding or dissociation of biomolecules on the surface of the biosensor will cause a change in the thickness of the film layer at the end of the sensor, thereby causing a change in the interference spectrum (c). By detecting the displacement of the interference spectrum in real time (Δλ), the binding and dissociation rates can be calculated, and the corresponding concentration changes can be obtained. BLI technology does not require labeling of other biomolecules, and directly detects the real-time interaction between two biomolecules, ensuring the authenticity of the experiment to the greatest extent.

Figure 1 Principle of BLI Technology

II. BLI Detection Method

2.1 Kinetic Test

The biomolecular interaction analysis system can detect the interaction between biomolecules in real time, and is widely used to determine the kinetic constants of proteins, nucleic acids and other biomolecules. It can provide reliable kinetic information including binding rate constant (Ka), dissociation rate constant (Kd) and affinity constant (KD). The steps of kinetic experiments mainly include the selection of biosensors, loading, quenching, baseline, association, dissociation, regeneration and neutralization.

2.2 Selection of Biosensors

The commonly used biosensors in kinetic constant determination experiments mainly include SA, SSA, AR2G, APS, AHC and AMC. Each biosensor is suitable for different biological samples, and the use requirements of different biosensors are also different. For example, SSA is only suitable for detecting small molecule samples; SA is used to solidify biotinylated proteins, antibodies, compounds and nucleic acids to detect macromolecular samples that interact with them; AHC can directly solidify human antibodies and then detect molecules that interact with them.

2.3 Solidification and Quenching

The quality of sample solidification directly affects the results of the test. When different biosensors are selected, the solidification method and conditions are also different. For example, DNA or polypeptides are usually first biotinylated and then solidified with SA; nanomaterials and liposomes are hydrophobically solidified with APS; polysaccharides are solidified on AR2G by amino coupling or solidified with SA after biotinylation. In addition, different biosensors have different requirements for curing conditions: SA needs to biotinylate the sample before curing; AR2G is very sensitive to the pH of curing, and the curing buffer used cannot contain any other protective agents with amino groups; the buffer of APS curing material must not contain any detergents. The curing concentration of the biosensor varies according to the type of sensor.

2.4 Binding and Dissociation

In order to obtain accurate kinetic constants, the buffer used for equilibrium, binding and dissociation in the experiment must be consistent. If you want to compare the dissociation under different conditions, you can consider performing the dissociation step in different buffers. If you don’t know the KD value, it is recommended to use a relatively high concentration sample or positive control before screening to first determine whether there is an interaction. If there is an interaction between the two samples, perform two rounds of screening.

III. Antigen-antibody Affinity Determination Process

The first step is to immerse the biosersor in a buffer solution for equilibrium; the second step is to immerse it in a known concentration of solidification solution, and the biotinylated antigen in the solution binds to the surface of the biosersor, increasing the thickness of the surface film layer; the third step is to immerse the biosersor with a known concentration of solidified antigen in a buffer solution as a baseline; the fourth step is to immerse the biosersor with a known concentration of solidified antigen in a sample solution containing the antibody to be tested, and the specific binding between antigen and antibody causes the thickness of the film layer to increase; the fifth step is to immerse the biosersor bound to the antibody to be tested in a buffer solution for dissociation, and the antibody to be tested falls off the surface of the biosersor, resulting in a decrease in the thickness of the film layer. The kinetic constant of the sample to be tested can be obtained by real-time monitoring of the biosersor biofilm thickness during the experiment.

Figure 2 Linear diagram of the detection process

IV. Main applications of BLI

Interactions between proteins: (1) Comprehensive analysis of affinity and kinetics of protein interactions, and study of protein functions, signal transduction and signal pathways in proteomics; (2) Screening of antibodies: Due to the rapid, label-free and real-time detection characteristics of biomembrane interference technology, the kinetic data obtained can be used for screening antibodies in antibody libraries or epitope mapping of polyclonal antibodies, especially for antibodies with low affinity or very fast dissociation; (3) Screening of small molecule compounds: It can quickly and accurately screen small molecule drugs that interact with target proteins; (4) Affinity and kinetics determination of antibodies and small molecule drugs; (5) Analysis of interactions between nucleic acids and proteins; (6) Interactions between virus particles and proteins.

TekBiotech provides label-free molecular interaction research based on BLI affinity detection technology: including protein-protein affinity detection; antigen-antibody affinity detection; protein and small molecule drug affinity detection; protein-DNA affinity detection; antibody subtype analysis; antibody pairing; hybridoma screening, etc., as well as affinity detection of low molecular weight compounds containing receptors and many other compounds and high-throughput affinity sorting services according to customer needs.